A : Effect of P2 receptor antagonism on ATP induced IL - 6 release . were treated with P2 - antagonist suramin ( 1 , 10 100µM ) alone or together with ATP ( 100µM ) for 24h .

O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O

A: Effect of P2 receptor antagonism on ATP induced IL-6 release. Cells were treated with P2-antagonist suramin (1, 10 and 100µM) alone or together with ATP (100µM) for 24h.


B : ATP induced IL - 6 release in msEGCs measured by ELISA . were treated with indicated concentrations of ATP - ATPɣS for 24h .

O O B-CONTROLLED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O

B: ATP induced IL-6 release in msEGCs measured by ELISA. Cells were treated with the indicated concentrations of ATP-and ATPɣS for 24h.


C : Effects of P2X antagonists on ATP induced IL - 6 release . were treated for 24h alone or together with ATP ( 100µM ) in absence or presence of P2X2 , P2X4 P2X7 - antagonists PSB - 1011 , 5 - BD - BD A740003 , respectively .

C: Effects of P2X antagonists on ATP induced IL-6 release. Cells were treated for 24h alone or together with ATP (100µM) in absence or presence of P2X2, P2X4 and P2X7-antagonists PSB-1011, 5-BD-BD and A740003, respectively.


D : P2X2 antagonism of ATP induced mRNA expression of IL - 6 , GFAP RCAN by qPCR in msEGCs . were treated with P2X2 - antagonist PSB - 1011 ( 20µM ) alone or together with ATP ( 10µM ) for 6h .

O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O

D: P2X2 antagonism of ATP induced mRNA expression of IL-6, GFAP and RCAN by qPCR in msEGCs. Cells were treated with the P2X2-antagonist PSB-1011 (20µM) alone or together with ATP (10µM) for 6h.


E : Representative confocal of P2X2 ( green ) GFAP ( violet ) positive msEGCs in vivo in vitro . arrows mark double positive ( ) . Scale bar 50µm .

O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O

E: Representative confocal images of P2X2 (green) and GFAP (violet) positive msEGCs in vivo and in vitro. White arrows mark double positive (white) cells. Scale bar 50µm.


F : P2X2 - siRNA reduces P2X2 - mRNA dampens gliosis gene expression after ATPɣS ( 100µM ) treatment for 6h .

O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O

F: P2X2-siRNA reduces P2X2-mRNA and dampens the gliosis gene expression after ATPɣS (100µM) treatment for 6h.


G : P2X2 - siRNA reduces IL - 6 release after ATPɣS treatment ( 10µM , 100µM ) for 6h .

O O B-CONTROLLED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-CONTROLLED_VAR O O O O O O O O O

G: P2X2-siRNA reduces IL-6 release after ATPɣS treatment (10µM, 100µM) for 6h.


A : ATP measurement at indicated postoperative time points in peritoneal lavages of mice that underwent intestinal manipulation ( IM ) or were naïve .

O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O

A: ATP measurement at the indicated postoperative time points in peritoneal lavages of mice that underwent intestinal manipulation (IM) or were naïve.


B : PCA plot of gene expression from POI mice at different disease stages naïve mice ; n = 3 for each group .

O O O O O O O O O O O O O O O O O O O O O O O O

B: PCA plot of gene expression from POI mice at different disease stages and naïve mice; n=3 for each group.


F - H : qPCR of indicated gliosis marker in mice that underwent IM ( n = 7 ) .

F-H: qPCR analysis of indicated gliosis marker in mice that underwent IM (n=7).


I : Histological of EGC proliferation in vivo . Representative confocal show Sox10 ( green ) Ki67 ( violet ) positive EGCs ( arrows ) in small bowel muscularis externa at naïve , IM6h 24h . Scale bar 50µm .

O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O

I: Histological analysis of EGC proliferation in vivo. Representative confocal images and quantification show Sox10 (green) and Ki67 (violet) positive EGCs (white arrows) in the small bowel muscularis externa at naïve, IM6h and 24h. Scale bar 50µm.


K - M : qPCR of indicated gliosis markers in td + glia from naïve mice mice that underwent IM ( n = 5 - 7 , POI mice ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

K-M: qPCR analysis of indicated gliosis markers in td+ glia cells from naïve mice and mice that underwent IM (n=5-7, POI mice).


Postoperative gene expression analyses of indicated gliosis marker in ME .

Postoperative gene expression analyses of indicated gliosis marker in the ME.


E : Histological of EGC proliferation by of Ki67 ( violet ) Sox10 positive EGCs at IM24h . Mice were treated as seen in ( A ) .

O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O

E: Histological analysis of EGC proliferation by quantification of Ki67 (violet) and Sox10 positive EGCs at IM24h. Mice were treated as seen in (A).


F : Histological counting of infiltrating myeloperoxidase ( MPO ) - positive leukocytes in postoperative ME ( n = 6 - 8 ) .

O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O

F: Histological counting of infiltrating myeloperoxidase (MPO)-positive leukocytes in the postoperative ME (n=6-8).


G : FACS of infiltrating in ME of mice treated with ambroxol or vehicle . CD45 , Ly6C CX3CR1 were used to distinguish resident macrophages ( CD45 + / Ly6C - / CX3CR1 + ) , infiltrating monocytes ( CD45 + / Ly6C + / CX3CR1 - ) infiltrated monocyte - derived macrophages ( CD45 + / Ly6C + / CX3CR1 + ) .

O O O O O O O O O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O B-MEASURED_VAR O O O

G: FACS analysis of infiltrating cells in the ME of mice treated with ambroxol or vehicle. CD45, Ly6C and CX3CR1 were used to distinguish resident macrophages (CD45+/Ly6C-/CX3CR1+), infiltrating monocytes (CD45+/Ly6C+/CX3CR1-) and infiltrated monocyte-derived macrophages (CD45+/Ly6C+/CX3CR1+).


H : Postoperative in vivo GI transit measurement in mice treated with ambroxol or vehicle .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O

H: Postoperative in vivo GI transit measurement in mice treated with ambroxol or vehicle.


E : IL - 6 release in hEGC cultures upon stimulation with ATP ( 200µM ) or ATPɣS ( 100µM ) after 6h 24h treatment ( n = 4 - 6 , hEGCs ) .

O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

E: IL-6 protein release in hEGC cultures upon stimulation with ATP (200µM) or ATPɣS (100µM) after 6h and 24h treatment (n=4-6, hEGCs).


F : IL - 6 release in hEGC cultures upon stimulation with NTPDase inhibitor ARL67156 at indicated concentrations ; n = 4 - 6 , hEGCs .

O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O

F: IL-6 protein release in hEGC cultures upon stimulation with NTPDase inhibitor ARL67156 at indicated concentrations; n=4-6, hEGCs.


G : Immunofluorescence microscopy revealed P2X2 expression ( green ) in a majority of s100β + ( violet ) hEGCs in intact myenteric ganglia of human colon . arrows mark double positive glia green arrows mark P2X2 positive neurons . Scale bar , 50µm .

O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O

G: Immunofluorescence microscopy revealed P2X2 expression (green) in a majority of s100β+ (violet) hEGCs in intact myenteric ganglia of the human colon. White arrows mark double positive glia cells and green arrows mark P2X2 positive neurons. Scale bar, 50µm.


H : Effect of P2X2 antagonism on ATP - induced IL - 6 release . ELISA measurement of IL - 6 in hEGCs upon treatment with P2X2 - antagonist PSB - 1011 ( 20µM ) alone or together with ATP ( 200µM ) treated for 24h ( n = 6 , hEGCs ) .

O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O

H: Effect of the P2X2 antagonism on ATP-induced IL-6 release. ELISA measurement of IL-6 in hEGCs upon treatment with P2X2-antagonist PSB-1011 (20µM) alone or together with ATP (200µM) treated for 24h (n=6, hEGCs).


I : PSB - 1011 ( 20μM ) treatment inhibited ATP - triggered calcium responses in HEK transfected with P2X2 . Data are represented as ΔF / F0 + ; n = 102 HEK . J : P2X2 receptor antagonist PSB - 0711 ( 20μM ) nearly abolished ATP - triggered calcium response in HEK transfected with P2X2 . n = 219 HEK .

O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O

I: PSB-1011 (20μM) treatment inhibited ATP-triggered calcium responses in HEK cells transfected with P2X2. Data are represented as ΔF/F0 +SEM; n=102 HEK cells. J: The P2X2 receptor antagonist PSB-0711 (20μM) nearly abolished the ATP-triggered calcium response in HEK cells transfected with P2X2. n=219 HEK cells.


K : Ambroxol blocks ATP - induced IL - 6 release in hEGCs ; release measurement by ELISA of IL - 6 in hEGCs . were treated with ATP ( 200 µM ) alone or together with ambroxol ( 20 µM ) for 24h ; n = 6 , hEGCs .

O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O

K: Ambroxol blocks ATP-induced IL-6 release in hEGCs; Protein release measurement by ELISA of IL-6 in hEGCs. Cells were treated with ATP (200 µM) alone or together with ambroxol (20 µM) for 24h; n=6, hEGCs.


L - N : qPCR of several gliosis ATP - target genes in mechanically manipulated surgical specimens incubated in presence or absence of ambroxol ( 20µM ) . ( n = 7 , 4 human ) .

O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O

L-N: qPCR analysis of several gliosis and ATP-target genes in the mechanically manipulated surgical specimens incubated in the presence or absence of ambroxol (20µM). (n=7, 4 human patients).


( C ) Fold induction of of CasTA candidates using different gRNAs targeting to different locations of GFP reporter gene compared to a without CasTA . dCas9 - ω was verified in ∆ rpoZ background , of candidates were verified in wild - type ( BW25113 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O

(C) Fold induction of the fluorescence of CasTA candidates using different gRNAs targeting to different locations of the GFP reporter gene compared to a strain without CasTA. The dCas9-ω was verified in the ∆rpoZ strain background, and the rest of candidates were verified in the wild-type strain (BW25113).


( D ) Survival of containing upregulated antibiotic resistance reporter induced by dCas9 or dCas9 - AsiA with gRNA - H3 under kanamycin selection ( 2 . 5 μg / ml ) .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O

(D) Survival of cells containing upregulated antibiotic resistance reporter induced by dCas9 or dCas9-AsiA with gRNA-H3 under kanamycin selection (2.5 μg/ml).


( E ) Different gRNAs were paired with dCas9 - AsiA dCas9 - ω to profile optimal gRNA binding . dCas9 - AsiA was verified in wild - type ( BW25113 ) , dCas9 - ω was examined in ∆ rpoZ background .

O O O O O O O O B-MEASURED_VAR O B-CONTROLLED_VAR B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O

(E) Different gRNAs were paired with dCas9-AsiA and dCas9-ω to profile the optimal gRNA binding distance. The dCas9-AsiA was verified in the wild-type strain (BW25113), and dCas9-ω was examined in the ∆rpoZ strain background.


( F ) Comparing single multiple gRNA with dCas9 - AsiA .

O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O

(F) Comparing single and multiple gRNA with dCas9-AsiA.


( C ) Distribution of signal of GFP reporter induced by different dCas9 - AsiA variants ( top ) . Fold induction by different dCas9 - AsiA variants is shown ( bottom ) .

O O O O O O O O O O O O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O

(C) Distribution of fluorescence signal of the GFP reporter induced by different dCas9-AsiA variants (top). Fold induction by different dCas9-AsiA variants is shown (bottom).


( D ) CRISPRa induction of promoters with varying basal expression . CRISPRa - , basal expression of promoter ; CRISPRa + , expression activated by dCas9 - AsiA - m2 . 1 associated gRNA - H4 .

O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O

(D) CRISPRa induction of promoters with varying basal expression levels. CRISPRa-, basal expression of the promoter; CRISPRa+, expression activated by dCas9-AsiA-m2.1 and associated gRNA-H4.


( A ) CasTA2 . 1 upregulates a genomically inserted GFP reporter .

O O O B-CONTROLLED_VAR O O O O O O O O O

(A) CasTA2.1 upregulates a genomically inserted GFP reporter.


( B ) Chromosomal gene targets activated by CasTA2 . 1 with bars showing activation fold change dots showing basal expression of each gene . Expression was quantified using RT - qPCR .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Chromosomal gene targets activated by CasTA2.1 with bars showing the activation fold change and dots showing basal expression of each gene. Expression was quantified using RT-qPCR.


( C ) CasTA2 . 1 can mediate CRISPRi with appropriate gRNA designs by positioning different gRNAs relative to target gene . A non - specific gRNA is used as negative control .

(C) CasTA2.1 can mediate CRISPRi with appropriate gRNA designs by positioning different gRNAs relative to the target gene. A non-specific gRNA is used as the negative control.


( D ) Demonstration of multiplexed CRISPRa CRISPRi using CasTA2 . 1 on a reporter containing GFP mScarlet . Parental had low basal GFP high basal mScarlet expression .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

(D) Demonstration of multiplexed CRISPRa and CRISPRi using CasTA2.1 on a reporter containing GFP and mScarlet. Parental cells had low basal GFP and high basal mScarlet expression.


( B ) Promoters significantly activated by CasTA2 . 1 using gRNA - H23 ( left ) or gRNA - H22 ( right ) are plotted with basal expression level on x - axis against fold activation by CRISPRa on y - axis . N is total number of promoters shown . Red box corresponds to highly activated promoters ( fold change > 10 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Promoters significantly activated by CasTA2.1 using gRNA-H23 (left) or gRNA-H22 (right) are plotted with basal expression level on x-axis against fold activation by CRISPRa on y-axis. N is the total number of promoters shown. Red box corresponds to highly activated promoters (fold change >10).


( C ) Highly activated promoters ( > 10 - fold ) using gRNA - H23 ( left ) or gRNA - H22 ( right ) are plotted . Basal expression ( purple lines ) , induced expression level ( orange lines on left or red lines on right , activated with gRNA - H23 or gRNA - H22 , respectively ) induced fold changes ( gray bars ) are shown . Promoters were first ranked grouped by basal expression . Within each group , promoters were ranked by activation fold changes .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Highly activated promoters (>10-fold) using gRNA-H23 (left) or gRNA-H22 (right) are plotted. Basal expression levels (purple lines), induced expression level (orange lines on left or red lines on right, activated with gRNA-H23 or gRNA-H22, respectively) and induced fold changes (gray bars) are shown. Promoters were first ranked and grouped by basal expression levels. Within each group, promoters were ranked by activation fold changes.


( B ) CRISPRi in S . enterica K . oxytoca using CasTA2 . 1 .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O

(B) CRISPRi in S. enterica and K. oxytoca using CasTA2.1.


( C ) CRISPRa in S . enterica K . oxytoca using CasTA1 . 0 ( dCas9 - AsiA _ wt ) , CasTA2 . 1 ( dCas9 - AsiA _ m2 . 1 ) or ancestral with basal promoter expression ( none ) .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O

(C) CRISPRa in S. enterica and K. oxytoca using CasTA1.0 (dCas9-AsiA_wt), CasTA2.1 (dCas9-AsiA_m2.1) or ancestral strain with basal promoter expression (none).


( B ) Western blotting of WT Drp1 - KO MEFs with or without FCCP treatment ( 30 min ) using indicated antibodies .

O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O

(B) Western blotting of WT and Drp1-KO MEFs with or without FCCP treatment (30 min) using the indicated antibodies.


( C ) of band . Values are average ± SD ( n = 3 ) . Significance was calculated using ANOVA with post - hoc Tukey : * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Quantification of band intensity. Values are average ± SD (n = 3). Significance was calculated using ANOVA with post-hoc Tukey: **p<0.01, ***p<0.001.


( B ) Western blotting of WT MEFs , Drp1 - KO MEFs , Drp1 - KO MEFs carrying Drp1 , of which express L2 - HA , using indicated antibodies . expression of L2 - HA was induced for 16 h ( 0 . 1 µg / ml doxycycline ) . asterisk indicates non - specific bands of anti - Oma1 antibodies .

O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O

(B) Western blotting of WT MEFs, Drp1-KO MEFs, and Drp1-KO MEFs carrying Drp1, all of which express L2-HA, using the indicated antibodies. The expression of L2-HA was induced for 16 h (0.1 µg/ml doxycycline). The asterisk indicates non-specific bands of anti-Oma1 antibodies.


( C ) of band . Values are average ± SD ( n = 3 ) .

(C) Quantification of band intensity. Values are average ± SD (n = 3).


( E ) WT Drp1 - KO MEFs , both of which express L2 - HA , were transduced with lentiviruses carrying either scramble or Oma1 - targeted shRNAs . Whole - lysates were analyzed by Western blotting using indicated antibodies . asterisk indicates non - specific bands of anti - Oma1 antibodies .

O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O

(E) WT and Drp1-KO MEFs, both of which express L2-HA, were transduced with lentiviruses carrying either scramble or Oma1-targeted shRNAs. Whole-cell lysates were analyzed by Western blotting using the indicated antibodies. The asterisk indicates non-specific bands of anti-Oma1 antibodies.


( F ) of band . Values are average ± SD ( n = 3 ) .

(F) Quantification of band intensity. Values are average ± SD (n = 3).


( A ) mitochondrial potential was measured in WT Drp1 - KO MEFs using a potential - dependent dye ( MitoLite NIR ) flow cytometry .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O

(A) The mitochondrial membrane potential was measured in WT and Drp1-KO MEFs using a membrane potential-dependent dye (MitoLite NIR) and flow cytometry.


( B ) Drp1 - KO MEFs carrying Su9 - GFP were treated with DMSO ( control ) or 10 nM antimycin A or 10 ng / ml oligomycin for 1 h viewed by scanning confocal microscopy for 30 min with 10 - s intervals in presence of TMRE . arrows indicate mitochondria that showed flickering . Three frames from time - lapse are shown ( please also see Movie EV1 - EV4 ) . Scale bar , 10 µm .

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Drp1-KO MEFs carrying Su9-GFP were treated with DMSO (control) or 10 nM antimycin A or 10 ng/ml oligomycin for 1 h and viewed by laser scanning confocal microscopy for 30 min with 10-s intervals in the presence of TMRE. The arrows indicate mitochondria that showed flickering. Three frames from the time-lapse analysis are shown (please also see Movie EV1-EV4). Scale bar, 10 µm.


of that showed flickering in 30 min . Values are average ± SD ( n = 3 experiments ) . In each experiment , 25 - 89 were analyzed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The percentage of cells that showed flickering in 30 min. Values are average ± SD (n = 3 experiments). In each experiment, 25-89 cells were analyzed.


( F ) Drp1 - KO MEFs were treated with DMSO or 10 nM antimycin A or 10 ng / ml oligomycin or 10 µM FCCP for 1 h . mitochondrial potential was analyzed using MitoLite NIR flow cytometry .

O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O

(F) Drp1-KO MEFs were treated with DMSO or 10 nM antimycin A or 10 ng/ml oligomycin or 10 µM FCCP for 1 h. The mitochondrial membrane potential was analyzed using MitoLite NIR and flow cytometry.


( G ) Drp1 - KO MEFs expressing L2 - HA ( induced for 16 h ) were treated with 10 nM antimycin A along with 10 µM FCCP . Western blotting was performed using indicated antibodies . asterisk indicates non - specific bands of anti - Oma1 antibodies . ( H ) of band . Values are average ± SD ( n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

(G) Drp1-KO MEFs expressing L2-HA (induced for 16 h) were treated with 10 nM antimycin A along with 10 µM FCCP. Western blotting was performed using the indicated antibodies. The asterisk indicates non-specific bands of anti-Oma1 antibodies. (H) Quantification of band intensity. Values are average ± SD (n = 3).


( G ) Drp1 - KO MEFs expressing L2 - HA ( induced for 16 h ) were treated with 10 nM antimycin A along with 10 µM FCCP . Western blotting was performed using indicated antibodies . asterisk indicates non - specific bands of anti - Oma1 antibodies .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O

(G) Drp1-KO MEFs expressing L2-HA (induced for 16 h) were treated with 10 nM antimycin A along with 10 µM FCCP. Western blotting was performed using the indicated antibodies. The asterisk indicates non-specific bands of anti-Oma1 antibodies.


( I ) Western blotting of Drp1 - KO MEFs with or without 10 ng / ml oligomycin treatment . asterisk indicates non - specific bands of anti - Oma1 antibodies . ( J ) of band . Values are average ± SD ( n = 3 ) .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

(I) Western blotting of Drp1-KO MEFs with or without 10 ng/ml oligomycin treatment. The asterisk indicates non-specific bands of anti-Oma1 antibodies. (J) Quantification of band intensity. Values are average ± SD (n = 3).


( I ) Western blotting of Drp1 - KO MEFs with or without 10 ng / ml oligomycin treatment . asterisk indicates non - specific bands of anti - Oma1 antibodies .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O O

(I) Western blotting of Drp1-KO MEFs with or without 10 ng/ml oligomycin treatment. The asterisk indicates non-specific bands of anti-Oma1 antibodies.


( K ) WT MEFs carrying Su9 - GFP were treated with DMSO or 10 ng / ml oligomycin for 1 h viewed by scanning confocal microscopy for 30 min with 10 - s intervals in presence of TMRE . arrows indicate mitochondria that showed flickering . Three frames from time - lapse are shown ( please also see Movie EV1 EV5 ) . Scale bar , 10 µm .

O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(K) WT MEFs carrying Su9-GFP were treated with DMSO or 10 ng/ml oligomycin for 1 h and viewed by laser scanning confocal microscopy for 30 min with 10-s intervals in the presence of TMRE. The arrows indicate mitochondria that showed flickering. Three frames from the time-lapse analysis are shown (please also see Movie EV1 and EV5). Scale bar, 10 µm.


( L ) WT MEFs expressing L2 - HA ( induced for 16 h ) were treated with 10 ng / ml oligomycin . Western blotting was performed using indicated antibodies . asterisk indicates non - specific bands of anti - Oma1 antibodies . ( M ) of band . Values are average ± SD ( n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

(L) WT MEFs expressing L2-HA (induced for 16 h) were treated with 10 ng/ml oligomycin. Western blotting was performed using the indicated antibodies. The asterisk indicates non-specific bands of anti-Oma1 antibodies. (M) Quantification of band intensity. Values are average ± SD (n = 3).


( L ) WT MEFs expressing L2 - HA ( induced for 16 h ) were treated with 10 ng / ml oligomycin . Western blotting was performed using indicated antibodies . asterisk indicates non - specific bands of anti - Oma1 antibodies .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O

(L) WT MEFs expressing L2-HA (induced for 16 h) were treated with 10 ng/ml oligomycin. Western blotting was performed using the indicated antibodies. The asterisk indicates non-specific bands of anti-Oma1 antibodies.


( A ) Western blotting of Drp1flox / floxOpa1flox / flox Drp1Opa1 - KO MEFs . asterisk indicates non - specific bands of anti - Oma1 antibodies . ( B ) of band . Values are average ± SD ( n = 3 ) .

O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

(A) Western blotting of Drp1flox/floxOpa1flox/flox and Drp1Opa1-KO MEFs. The asterisk indicates non-specific bands of anti-Oma1 antibodies. (B) Quantification of band intensity. Values are average ± SD (n = 3).


( A ) Western blotting of Drp1flox / floxOpa1flox / flox Drp1Opa1 - KO MEFs . asterisk indicates non - specific bands of anti - Oma1 antibodies .

O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O

(A) Western blotting of Drp1flox/floxOpa1flox/flox and Drp1Opa1-KO MEFs. The asterisk indicates non-specific bands of anti-Oma1 antibodies.


( C ) Drp1flox / floxOpa1flox / flox MEFs Drp1Opa1 - KO MEFs were transduced with indicated Opa1 constructs . were observed using scanning confocal microscopy for 30 min with 10 - s intervals in presence of TMRE . of that exhibited flickering is shown . Values are average ± SD ( n = 3 experiments ) . In each experiment , 48 - 62 were analyzed .

O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Drp1flox/floxOpa1flox/flox MEFs and Drp1Opa1-KO MEFs were transduced with the indicated Opa1 constructs. The cells were observed using laser scanning confocal microscopy for 30 min with 10-s intervals in the presence of TMRE. The percentage of cells that exhibited flickering is shown. Values are average ± SD (n = 3 experiments). In each experiment, 48-62 cells were analyzed.


( D ) Drp1flox / floxOpa1flox / flox MEFs Drp1Opa1 - KO MEFs carrying indicated Opa1 constructs were subjected to confocal immunofluorescence microscopy using anti - PDH antibodies . Scale bar , 10 µm .

O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

(D) Drp1flox/floxOpa1flox/flox MEFs and Drp1Opa1-KO MEFs carrying the indicated Opa1 constructs were subjected to confocal immunofluorescence microscopy using anti-PDH antibodies. Scale bar, 10 µm.


( E ) of mitochondrial is shown ( n = 3 experiments ) . In each experiment , 30 were analyzed .

O O O O O O O O O O O O O O O O O O O O O O

(E) Quantification of mitochondrial morphology is shown (n = 3 experiments). In each experiment, 30 cells were analyzed.


( G ) Western blotting of Drp1flox / floxOpa1flox / flox MEFs Drp1Opa1 - KO MEFs , both of which carry indicated Opa1 constructs . asterisk indicates non - specific bands of anti - Oma1 antibodies .

O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O

(G) Western blotting of Drp1flox/floxOpa1flox/flox MEFs and Drp1Opa1-KO MEFs, both of which carry the indicated Opa1 constructs. The asterisk indicates non-specific bands of anti-Oma1 antibodies.


( H ) of band . Values are average ± SD ( n = 3 ) .

(H) Quantification of band intensity. Values are average ± SD (n = 3).


( B ) Three frames from time - lapse during artificial flickering are shown . boxed regions are magnified . Scale bar , 5 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Three frames from the time-lapse analysis during artificial flickering are shown. The boxed regions are magnified. Scale bar, 5 µm.


( C ) of Su9 - GFP TMRE in mitochondria was quantified .

(C) The fluorescence intensity of Su9-GFP and TMRE in mitochondria was quantified.


( D ) Western blotting of WT MEFs after indicated FCCP treatments . asterisk indicates non - specific bands of anti - Oma1 antibodies . ( E ) of band . Values are average ± SD ( n = 3 ) . Significance was calculated using ANOVA with post - hoc Tukey : * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Western blotting of WT MEFs after the indicated FCCP treatments. The asterisk indicates non-specific bands of anti-Oma1 antibodies. (E) Quantification of band intensity. Values are average ± SD (n = 3). Significance was calculated using ANOVA with post-hoc Tukey: **p<0.01, ***p<0.001.


( D ) Western blotting of WT MEFs after indicated FCCP treatments . asterisk indicates non - specific bands of anti - Oma1 antibodies .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O O

(D) Western blotting of WT MEFs after the indicated FCCP treatments. The asterisk indicates non-specific bands of anti-Oma1 antibodies.


( A ) Western blotting of WT MEFs Drp1 - KO MEFs , both of which carry shRNAs ( Oma1 or scramble ) / or ectopic Mfn1 . asterisk indicates non - specific bands of anti - Oma1 antibodies . ( B ) of band is shown . Values are average ± SD ( n = 3 ) .

O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O

(A) Western blotting of WT MEFs and Drp1-KO MEFs, both of which carry shRNAs (Oma1 or scramble) and/or ectopic Mfn1. The asterisk indicates non-specific bands of anti-Oma1 antibodies. (B) Quantification of band intensity is shown. Values are average ± SD (n = 3).


( A ) Western blotting of WT MEFs Drp1 - KO MEFs , both of which carry shRNAs ( Oma1 or scramble ) / or ectopic Mfn1 . asterisk indicates non - specific bands of anti - Oma1 antibodies .

O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O

(A) Western blotting of WT MEFs and Drp1-KO MEFs, both of which carry shRNAs (Oma1 or scramble) and/or ectopic Mfn1. The asterisk indicates non-specific bands of anti-Oma1 antibodies.


( C - F ) WT MEFs ( C E ) Drp1 - KO MEFs ( D F ) were transduced with Su9 - Eos , along with Oma1 shRNAs ectopic Mfn1 . Su9 - Eos in small region of mitochondria ( indicated by asterisks ) was photoconverted using a 405 - nm on confocal microscope . Within 1 s after photoconversion , were obtained for both photoconverted unconverted Su9 - Eos signals . before photoconversion were also taken to detect background signals . Scale bar , 10 µm . ( E F ) To determine connectivity of mitochondria , containing photoconverted Su9 - Eos signals was divided by containing photoconverted unconverted Su9 - Eos signals ( total mitochondria ) . Values are average ± SD ( n = 30 ) .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

(C-F) WT MEFs (C and E) and Drp1-KO MEFs (D and F) were transduced with Su9-Eos, along with Oma1 shRNAs and ectopic Mfn1. Su9-Eos in the small region of mitochondria (indicated by the asterisks) was photoconverted using a 405-nm laser on confocal microscope. Within 1 s after photoconversion, images were obtained for both photoconverted and unconverted Su9-Eos signals. Images before the photoconversion were also taken to detect background signals. Scale bar, 10 µm. (E and F) To determine the connectivity of mitochondria, the area containing photoconverted Su9-Eos signals was divided by the area containing the photoconverted and unconverted Su9-Eos signals (total mitochondria). Values are average ± SD (n = 30 cells).


WT which carry shRNAs ( Oma1 or scramble ) ectopic Mfn1 , were stained with TMRE . of that maintained potential is shown . Values are average ± SD ( n = 3 ) . 100 - 150 were analyzed in each experiment .

O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

WT which carry shRNAs (Oma1 or scramble) and ectopic Mfn1, were stained with TMRE. The percentage of cells that maintained the membrane potential is shown. Values are average ± SD (n = 3). 100-150 cells were analyzed in each experiment.


Drp1 - KO MEFs which carry shRNAs ( Oma1 or scramble ) ectopic Mfn1 , were stained with TMRE . arrows indicate that lost mitochondrial potential in ( C ) . Scale bar , 10 µm . of that maintained potential is shown . Values are average ± SD ( n = 3 ) . 100 - 150 were analyzed in each experiment .

B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Drp1-KO MEFs which carry shRNAs (Oma1 or scramble) and ectopic Mfn1, were stained with TMRE. The arrows indicate cells that lost the mitochondrial membrane potential in (C). Scale bar, 10 µm. The percentage of cells that maintained the membrane potential is shown. Values are average ± SD (n = 3). 100-150 cells were analyzed in each experiment.


OCRs were analyzed in same set of MEFs . of basal OCRs respiratory capacity is shown . Values are average ± SD ( n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O

OCRs were analyzed in the same set of MEFs. Quantification of the basal OCRs and respiratory capacity is shown. Values are average ± SD (n = 3).


( A ) Western blotting of livers isolated from indicated mice at 3 months of using indicated antibodies . ( B ) of band . Values are average ± SD ( n = 3 mice for each genotype ) . Significance was calculated using ANOVA with post - hoc Tukey : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Western blotting of livers isolated from the indicated mice at 3 months of age using the indicated antibodies. (B) Quantification of band intensity. Values are average ± SD (n = 3 mice for each genotype). Significance was calculated using ANOVA with post-hoc Tukey: *p<0.05, **p<0.01, ***p<0.001.


( A ) HEK293A lines stably expressing GFP , GFP - WIPI1a , GFP - WIPI2b were incubated in full medium ( F ) or starvation medium ( EBSS ) ( S ) for 2 hr before being used for GFP - TRAP pull - down .

O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) HEK293A cell lines stably expressing GFP, GFP-WIPI1a, and GFP-WIPI2b were incubated in full medium (F) or starvation medium (EBSS) (S) for 2 hr before being used for GFP-TRAP pull-down.


( B ) WIPI2 was immunoprecipitated from HEK293A treated with DSP , at indicated concentrations , before immunoblotting .

O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O

(B) WIPI2 was immunoprecipitated from HEK293A cells treated with DSP, at the indicated concentrations, before immunoblotting.


( C ) WT or Atg16L1Δ / Δ MEFs in fed medium or starved for 2 hr in EBSS were fixed labeled with an anti - WIPI2 antibody . Scale bars , 10 μm . ( D ) WIPI2 in ( C ) were counted , a statistical of WIPI2 was performed using an unpaired Student ' s t . ∗ p < 0 . 05 . for n = 3 .

O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O

(C) WT or Atg16L1Δ/Δ MEFs in fed medium or starved for 2 hr in EBSS were fixed and labeled with an anti-WIPI2 antibody. Scale bars, 10 μm.(D) WIPI2 puncta in (C) were counted, and a statistical analysis of WIPI2 puncta was performed using an unpaired Student's t test. ∗p < 0.05. SEM for n = 3.


( E - H ) CLEM ( correlative electron microscopy ) of endogenous WIPI2 in Atg16L1Δ / Δ MEFs . ( E ) Merged phase confocal section of Atg16L1Δ / Δ labeled with anti - WIPI2 antibody . Scale bar , 10 μm . ( F ) Low - magnification of in ( E ) . Scale bar 10μm . ( G H ) High magnification of boxed regions in ( E ) ( F ) . Top box bottom box indicate panels ( G ) ( H ) , respectively . Arrows indicate open phagophores . M , mitochondria ; ER , endoplasmic reticulum . Scale bars , 1 μm ( G ) 0 . 5 μm ( H ) .

O O O O O O O O O O O O O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E-H) CLEM (correlative light and electron microscopy) of endogenous WIPI2 in Atg16L1Δ/Δ MEFs.(E) Merged phase and confocal section of Atg16L1Δ/Δ cells labeled with anti-WIPI2 antibody. Scale bar, 10 μm.(F) Low-magnification TEM of cell in (E). Scale bar 10μm.(G and H) High magnification of boxed regions in (E) and (F). Top box and bottom box indicate panels (G) and (H), respectively. Arrows indicate open phagophores. M, mitochondria; ER, endoplasmic reticulum. Scale bars, 1 μm (G) and 0.5 μm (H).


( I J ) 2GL9 ( GFP - LC3 HEK293 ; Chan et al . , 2007 ) were starved for 2 hr before visualization using indicated antibodies . Scale bars , 10 μm .

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

(I and J) 2GL9 cells (GFP-LC3 HEK293 cells; Chan et al., 2007) were starved for 2 hr before visualization using indicated antibodies. Scale bars, 10 μm.


( K ) Live - imaging demonstrates that Atg16L1 translocates to WIPI2b - positive in starvation . HEK293 expressing GFP - WIPI2b mCherry - Atg16L1 were starved in EBSS imaged every 2 s using a spinning disk microscope . See also Figure S1 Movie S1 .

O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O

(K) Live-cell imaging demonstrates that Atg16L1 translocates to WIPI2b-positive puncta in starvation. HEK293 cells expressing GFP-WIPI2b and mCherry-Atg16L1 were starved in EBSS and imaged every 2 s using a spinning disk microscope. See also Figure S1 and Movie S1.


( B ) Untransfected ( UN ) or FLAG - Atg16L1 full - ( FL ) , 79 - 623 , or 1 - 265 constructs were expressed in HEK293A stably expressing GFP - WIPI2b immunoprecipitated using GFP - TRAP . Tags were visualized by immunoblotting . I , input ; UB , unbound .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O

(B) Untransfected (UN) or FLAG-Atg16L1 full-length (FL), 79-623, or 1-265 constructs were expressed in HEK293A cells stably expressing GFP-WIPI2b and immunoprecipitated using GFP-TRAP. Tags were visualized by immunoblotting. I, input; UB, unbound.


( C ) GFP , CFP - Atg5 , GFP - WIPI1a , GFP - WIPI2b were transiently expressed in HEK293A . GFP - tagged were isolated using GFP - TRAP incubated with in vitro translated 35S - labeled FLAG - Atg16L1 constructs 1 - 265 , 1 - 242 , 1 - 230 , 1 - 207 before washing by autoradiography . expression was validated by immunoblot ( bottom panel ) .

O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) GFP, CFP-Atg5, GFP-WIPI1a, and GFP-WIPI2b were transiently expressed in HEK293A cells. GFP-tagged proteins were isolated using GFP-TRAP and incubated with in vitro translated 35S-labeled FLAG-Atg16L1 constructs 1-265, 1-242, 1-230, and 1-207 before washing and analysis by autoradiography. Protein expression was validated by immunoblot (bottom panel).


( D ) Lysates from HEK293A transiently expressing GFP , GFP - WIPI1a , or GFP - WIPI2b were mixed with lysates from HEK293A transiently expressing FLAG - Atg16L1 or Atg16L2 . complexes were immunoprecipitated using GFP - TRAP , followed by immunoblot . See also Figure S2 Table S1 .

O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O

(D) Lysates from HEK293A cells transiently expressing GFP, GFP-WIPI1a, or GFP-WIPI2b were mixed with lysates from HEK293A cells transiently expressing FLAG-Atg16L1 or Atg16L2. Protein complexes were immunoprecipitated using GFP-TRAP, followed by immunoblot. See also Figure S2 and Table S1.


( B ) Lysates from HEK293A transfected with GFP - WIPI2b were mixed with lysates from HEK293A transfected with FLAG - Atg16L2 , FLAG - Atg16L1 , or Flag - Atg16L1 mutants , immunoprecipitated , analyzed by immunoblotting . ( C ) Statistical of FLAG - Atg16L1 binding in ( B ) was performed by Student ' s t . for n = 4 . ∗ p < 0 . 05 .

O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Lysates from HEK293A cells transfected with GFP-WIPI2b were mixed with lysates from HEK293A cells transfected with FLAG-Atg16L2, FLAG-Atg16L1, or Flag-Atg16L1 mutants, immunoprecipitated, and analyzed by immunoblotting.(C) Statistical analysis of FLAG-Atg16L1 binding in (B) was performed by Student's t test. SEM for n = 4. ∗p < 0.05.


( D ) lysates from HEK293A transiently expressing FLAG - Atg16L2 , FLAG - Atg16L1 , or FLAG - Atg16L1 mutants were subjected to immunoprecipitation using FLAG M2 agarose beads . complexes were analyzed using immunoblotting . ( E ) Statistical of FIP200 binding in ( D ) was performed by Student ' s t . for n = 3 . ∗ p < 0 . 05 .

O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Cell lysates from HEK293A cells transiently expressing FLAG-Atg16L2, FLAG-Atg16L1, or FLAG-Atg16L1 mutants were subjected to immunoprecipitation using FLAG M2 agarose beads. Protein complexes were analyzed using immunoblotting.(E) Statistical analysis of FIP200 binding in (D) was performed by Student's t test. SEM for n = 3. ∗p < 0.05.


( F ) WIPI2 was immunoprecipitated from lysates from WT or FIP200 − / − MEFS after treatment with 0 . 5 mM DSP . Bound Atg16 Atg12 - 5 were detected by immunoblotting .

O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O

(F) WIPI2 was immunoprecipitated from lysates from WT or FIP200−/− MEFS after treatment with 0.5 mM DSP. Bound Atg16 and Atg12-5 were detected by immunoblotting.


( G ) HEK293 stably expressing GFP - WIPI2b were transiently transfected either FLAG - Atg16L1 WT , E226R E230R ( ERER ) , or D237R D239R ( DRDR ) . Bound input ( bottom ) were analyzed by immunoblotting .

O O O O O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

(G) HEK293 cells stably expressing GFP-WIPI2b were and transiently transfected either FLAG-Atg16L1 WT, E226R E230R (ERER), or D237R D239R (DRDR). Bound and input (bottom) were analyzed by immunoblotting.


( A ) Lysates from HEK293A transiently expressing GFP , GFP - WIPI1a , GFP - WIPI2a , GFP - WIPI2b , or GFP - WIPI2b FTTG mutant were used for GFP - Trap . Endogenous Atg16L1 binding was analyzed by immunoblot . ( B ) Statistical of ( A ) was performed by one - way ANOVA with Tukey ' s posttest . for n = 3 . ∗ p < 0 . 05 .

O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Lysates from HEK293A cells transiently expressing GFP, GFP-WIPI1a, GFP-WIPI2a, GFP-WIPI2b, or GFP-WIPI2b FTTG mutant were used for GFP-Trap. Endogenous Atg16L1 binding was analyzed by immunoblot.(B) Statistical analysis of (A) was performed by one-way ANOVA with Tukey's posttest. SEM for n = 3. ∗p < 0.05.


( D ) GFP - Trap from HEK293A transiently expressing GFP , GFP - WIPI1a , GFP - WIPI2b , GFP - WIPI2b R108E , GFP - WIPI2b R125E , or GFP - WIPI2b R108E R125E was mixed with in vitro translated 35S - labeled FLAG - Atg16L1 analyzed by autoradiography .

O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O

(D) GFP-Trap from HEK293A cells transiently expressing GFP, GFP-WIPI1a, GFP-WIPI2b, GFP-WIPI2b R108E, GFP-WIPI2b R125E, or GFP-WIPI2b R108E R125E was mixed with in vitro translated 35S-labeled FLAG-Atg16L1 and analyzed by autoradiography.


( E ) Lysates from HEK293A transiently expressing GFP , GFP - WIPI2b , GFP - WIPI2b ΔCT , GFP - WIPI2a , GFP - WIPI1a , or GFP - WIPI1a ΔCT were used for GFP - Trap . Endogenous Atg16L1 binding was analyzed by immunoblot . ( F ) Statistical of ( E ) was performed by one - way ANOVA with Tukey ' s posttest . for n = 2 . ∗ p < 0 . 05 .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Lysates from HEK293A cells transiently expressing GFP, GFP-WIPI2b, GFP-WIPI2b ΔCT, GFP-WIPI2a, GFP-WIPI1a, or GFP-WIPI1a ΔCT were used for GFP-Trap. Endogenous Atg16L1 binding was analyzed by immunoblot.(F) Statistical analysis of (E) was performed by one-way ANOVA with Tukey's posttest. SEM for n = 2. ∗p < 0.05.


( G ) Lysates from HEK293A transiently expressing GFP - WIPI2b WT , GFP - WIPI2b R108E , R125E , or R108E R125E were mixed with lysates from HEK293A transiently expressing FLAG - Atg16L1 WT , E226R , E230R , or E226R E230R in possible permutations . complexes from mixed lysates were immunoprecipitated using GFP - Trap , followed by immunoblot . ( H ) Statistical of ( G ) was performed by one - way ANOVA with Tukey ' s posttest . from n = 3 . ∗ p < 0 . 05 .

O O O O O O O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Lysates from HEK293A cells transiently expressing GFP-WIPI2b WT, GFP-WIPI2b R108E, R125E, or R108E R125E were mixed with lysates from HEK293A cells transiently expressing FLAG-Atg16L1 WT, E226R, E230R, or E226R E230R in all possible permutations. Protein complexes from mixed lysates were immunoprecipitated using GFP-Trap, followed by immunoblot analysis.(H) Statistical analysis of (G) was performed by one-way ANOVA with Tukey's posttest. SEM from n = 3. ∗p < 0.05.


( I ) CLEM of HEK293 treated with siRNA to WIPI2 were transfected with GFP - WIPI2b RERE starved in EBSS . Bright - field image ( top ) , GFP - WIPI2 RERE signal ( middle ) , of selected ( bottom ) . Boxed shown in ( J ) . Scale bars represent 10 μM for bright - field confocal 5 μM for . ( J ) High - magnification of boxed in ( I ) . Arrows indicate open phagophores in vicinity of ER mitochondria ( M ) , which contain GFP - WIPI2b RERE . See also Figures S4 S5 Table S1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O

(I) CLEM analysis of HEK293 cells treated with siRNA to WIPI2 were transfected with GFP-WIPI2b RERE and starved in EBSS. Bright-field image (top), GFP-WIPI2 RERE signal (middle), TEM of selected cell (bottom). Boxed area shown in (J). Scale bars represent 10 μM for bright-field and confocal and 5 μM for TEM.(J) High-magnification TEM of boxed area in (I). Arrows indicate open phagophores in the vicinity of ER and mitochondria (M), which contain GFP-WIPI2b RERE. See also Figures S4 and S5 and Table S1.


( A ) GFP , siRNA - resistant GFP - WIPI2b , or GFP - WIPI2b RERE was expressed in HEK293A treated for 72 hr with either RISC - free ( RF ) or WIPI2 siRNA . were left in full medium ( F ) or starved for 2 hr with EBSS ( S ) or EBSS with BafA ( B ) before immunoblot . ( B ) Statistical of ( A ) . for n = 3 . Statistical was performed by one - way ANOVA with Tukey ' s posttest . ∗ p < 0 . 05 .

O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) GFP, siRNA-resistant GFP-WIPI2b, or GFP-WIPI2b RERE was expressed in HEK293A cells treated for 72 hr with either RISC-free (RF) or WIPI2 siRNA. Cells were left in full medium (F) or starved for 2 hr with EBSS (S) or EBSS with BafA (B) before immunoblot analysis.(B) Statistical analysis of (A). SEM for n = 3. Statistical analysis was performed by one-way ANOVA with Tukey's posttest. ∗p < 0.05.


( C ) GFP , siRNA - resistant GFP - WIPI2b , GFP - WIPI2b RERE , GFP - WIPI2b FTTG , or GFP - WIPI2b FTTG RERE was expressed in HEK293A treated for 72 hr with WIPI2 siRNA . were left in full medium ( F ) or starved for 2 hr with EBSS ( S ) or EBSS with BafA ( B ) before immunoblot . ( D ) Statistical of ( C ) was performed by one - way ANOVA with Tukey ' s posttest . for LC3 ( n = 2 ) p62 ( n = 4 ) are shown . ∗ p < 0 . 05 .

O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O

(C) GFP, siRNA-resistant GFP-WIPI2b, GFP-WIPI2b RERE, GFP-WIPI2b FTTG, or GFP-WIPI2b FTTG RERE was expressed in HEK293A cells treated for 72 hr with WIPI2 siRNA. Cells were left in full medium (F) or starved for 2 hr with EBSS (S) or EBSS with BafA (B) before immunoblot analysis.(D) Statistical analysis of (C) was performed by one-way ANOVA with Tukey's posttest. The SEM for LC3 (n = 2) and p62 (n = 4) are shown. ∗p < 0.05.


( E ) siRNA - resistant GFP - WIPI2b , GFP - WIPI2b RERE , GFP - WIPI2b FTTG , or GFP - WIPI2b FTTG RERE was expressed in HEK293A treated for 72 hr with WIPI2 siRNA . were starved in EBSS for 2 hr without or with BafA , fixed , labeled , LC3 was visualized by confocal microscopy . Scale bars , 10 μm . ( F ) of WIPI2 per from ( E ) . from 10 per condition ; ∗ ∗ ∗ p < 0 . 001 using one - way ANOVA . ( G ) Statistical of LC3 from ( E ) with GFP control ( not shown in E ) . for n = 3 . Statistical was performed by one - way ANOVA with Dunn ' s posttest t . ∗ p < 0 . 05 . See also Table S1 .

O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) siRNA-resistant GFP-WIPI2b, GFP-WIPI2b RERE, GFP-WIPI2b FTTG, or GFP-WIPI2b FTTG RERE was expressed in HEK293A cells treated for 72 hr with WIPI2 siRNA. Cells were starved in EBSS for 2 hr without or with BafA, fixed, and labeled, and LC3 was visualized by confocal microscopy. Scale bars, 10 μm.(F) Quantification of WIPI2 puncta per cell from (E). SEM from 10 cells per condition; ∗∗∗p < 0.001 using one-way ANOVA.(G) Statistical analysis of LC3 puncta from (E) with GFP control (not shown in E). SEM for n = 3. Statistical analysis was performed by one-way ANOVA with Dunn's posttest t test. ∗p < 0.05. See also Table S1.


( A ) HEK293A transiently expressing indicated mCherry constructs were treated in either full medium ( F ) , starvation medium ( S ) , or starvation medium with wortmannin ( W ) for 2 hr before immunoblot . ( B ) Statistical of ( A ) was performed by one - way ANOVA with Tukey ' s post hoc . for n = 3 . ∗ p < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) HEK293A cells transiently expressing the indicated mCherry constructs were treated in either full medium (F), starvation medium (S), or starvation medium with wortmannin (W) for 2 hr before immunoblot analysis.(B) Statistical analysis of (A) was performed by one-way ANOVA with Tukey's post hoc test. SEM for n = 3. ∗p < 0.05.


( C ) Complexes from HEK293A transiently expressing indicated mCherry constructs were immunoprecipitated using RFP - Trap before by immunoblotting .

O O O O O O O O O O O O O O O O O O O O O

(C) Complexes from HEK293A cells transiently expressing the indicated mCherry constructs were immunoprecipitated using RFP-Trap before analysis by immunoblotting.


( D ) 2GL9 transiently expressing indicated mCherry constructs treated as in ( A ) were fixed visualized by confocal microscopy .

O O O O O O O O O O O O O O O O O O O O O O

(D) 2GL9 cells transiently expressing the indicated mCherry constructs treated as in (A) were fixed and visualized by confocal microscopy.


( E ) CLEM of MCF7 expressing GFP - LC3 mCherry - WIPI2b CAAX . Bright - field confocal image merged ( left ) , confocal of expressing ( middle ) , low - magnification of of interest ( right ) . Boxed indicates plasma region showing colocalization of GFP - LC3 mCherry - WIPI2b . Scale bar , 10 μm ( n = 3 ) . ( F ) High - magnification showing plasma region boxed in ( E ) . Magnified insets , in top right , show small vesicular clusters under plasma detected in mCherry - WIPI2b CAAX , GFP - LC3 expressing but not in untransfected from a control experiment .

O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O

(E) CLEM of MCF7 cells expressing GFP-LC3 and mCherry-WIPI2b CAAX. Bright-field and confocal image merged (left), confocal of expressing cell (middle), low-magnification TEM of cell of interest (right). Boxed area indicates plasma membrane region showing colocalization of GFP-LC3 and mCherry-WIPI2b. Scale bar, 10 μm (n = 3).(F) High-magnification TEM showing plasma membrane region boxed in (E). Magnified insets, in top right, show small vesicular clusters under the plasma membrane detected in mCherry-WIPI2b CAAX, GFP-LC3 expressing cells but not in untransfected cells from a control experiment.


( G ) HEK293A treated with either RISC - free ( RF ) , Atg16L1 siRNA , or FIP200 siRNA for 72 hr before transfection with HA - WIPI2b - CAAX were incubated in full medium ( F ) , EBSS ( S ) , or EBSS with wortmannin ( W ) for 2 hr before immunoblot . Please note that WIPI2 constructs are FTTG mutants . See also Figure S6 Table S1 .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O

(G) HEK293A cells treated with either RISC-free (RF), Atg16L1 siRNA, or FIP200 siRNA for 72 hr before transfection with HA-WIPI2b-CAAX were incubated in full medium (F), EBSS (S), or EBSS with wortmannin (W) for 2 hr before immunoblot analysis. Please note that all WIPI2 constructs are FTTG mutants. See also Figure S6 and Table S1.


( A ) Atg16L1Δ / Δ MEFs were transiently transfected with either mock , FLAG - tagged Atg16L1 WT , ERER , or DRDR . After 2 hr in EBSS , were fixed labeled with anti - LC3 anti - FLAG antibodies visualized by confocal microscopy . Scale bars , 20 μm . ( B ) Statistical of ( A ) was performed using one - way ANOVA with Dunn ' s posttest . ∗ p < 0 . 05 . for n = 3 .

O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Atg16L1Δ/Δ MEFs were transiently transfected with either mock, FLAG-tagged Atg16L1 WT, ERER, or DRDR. After 2 hr in EBSS, cells were fixed and labeled with anti-LC3 and anti-FLAG antibodies and visualized by confocal microscopy. Scale bars, 20 μm.(B) Statistical analysis of (A) was performed using one-way ANOVA with Dunn's posttest. ∗p < 0.05. SEM for n = 3.


( C ) Atg16L1Δ / Δ MEFs were transiently transfected with either mock , FLAG - tagged Atg16L1 WT , E226R E230R ( ERER ) , or D237R D239R ( DRDR ) before by immunoblotting . ( D ) Statistical of ( C ) was performed by one - way ANOVA with Tukey ' s post hoc . for n = 3 . ∗ p < 0 . 05 .

O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Atg16L1Δ/Δ MEFs were transiently transfected with either mock, FLAG-tagged Atg16L1 WT, E226R E230R (ERER), or D237R D239R (DRDR) before analysis by immunoblotting.(D) Statistical analysis of (C) was performed by one-way ANOVA with Tukey's post hoc test. SEM for n = 3. ∗p < 0.05.


( E ) HEK293A were treated with either RISC - free control or WIPI2 siRNA before infection with Salmonella ( moi = 100 ) for 1 hr , labeled with anti - LC3 , WIPI2 , p62 antibodies , followed by confocal . Scale bars , 5 μm . ( F ) Statistical for ( E ) was performed using an unpaired Student ' s t . ∗ p < 0 . 05 . for n = 3 .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) HEK293A cells were treated with either RISC-free control or WIPI2 siRNA before infection with Salmonella (moi = 100) for 1 hr, labeled with anti-LC3, WIPI2, and p62 antibodies, and followed by confocal analysis. Scale bars, 5 μm.(F) Statistical analysis for (E) was performed using an unpaired Student's t test. ∗p < 0.05. SEM for n = 3.


( G ) Atg16L1Δ / Δ MEFs were transiently transfected with FLAG - Atg16L1 WT , FLAG - Atg16L1 ERER , or FLAG - Atg16L2 DRDR 24 hr before being infected with Salmonella ( moi = 25 ) for 1 hr , labeled with anti - LC3 , anti - p62 , anti - FLAG antibodies , followed by by confocal microscopy . Scale bars , 5 μm . ( H ) Statistical of ( G ) was performed by one - way ANOVA with Dunn ' s post hoc . for n = 4 . ∗ p < 0 . 05 . See also Figure S7 .

O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Atg16L1Δ/Δ MEFs were transiently transfected with FLAG-Atg16L1 WT, FLAG-Atg16L1 ERER, or FLAG-Atg16L2 DRDR 24 hr before being infected with Salmonella (moi = 25) for 1 hr, labeled with anti-LC3, anti-p62, and anti-FLAG antibodies, and followed by analysis by confocal microscopy. Scale bars, 5 μm.(H) Statistical analysis of (G) was performed by one-way ANOVA with Dunn's post hoc test. SEM for n = 4. ∗p < 0.05. See also Figure S7.


A RT - qPCR reveals that miR - 210 was increased in lungs of mice with PH triggered by various conditions : VHL − / − as compared with VHL + / + mice ( N = 4 / group ) , * * * P < 0 . 0001 ( first graph ) ; hypoxia + SU5416 ( Hyp + SU5416 ) ( N = 6 / group ) as compared with normoxia + SU5416 ( Norm + SU5416 ) ( N = 7 / group ) , * * P = 0 . 0015 ( second graph ) ; Il6 transgenic versus littermate control mice ( N = 4 / group ) , * * P = 0 . 0097 ( third graph ) ; S . mansoni - infected mice ( N = 4 ) compared with non - infected control mice ( N = 5 ) , * * * P < 0 . 0001 ( fourth graph ) .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A RT-qPCR reveals that miR-210 was increased in lungs of mice with PH triggered by various conditions: VHL−/− as compared with VHL+/+ mice (N = 4/group), ***P < 0.0001 (first graph); hypoxia + SU5416 (Hyp + SU5416) (N = 6/group) as compared with normoxia + SU5416 (Norm + SU5416) (N = 7/group), **P = 0.0015 (second graph); Il6 transgenic versus littermate control mice (N = 4/group), **P = 0.0097 (third graph); and S. mansoni-infected mice (N = 4) compared with non-infected control mice (N = 5), ***P < 0.0001 (fourth graph).


B From subjects in ( A ) , in situ hybridization ( ISH , purple stain ) revealed increased miR - 210 in < 100 - μm pulmonary vessels of mice suffering from PH , * P = 0 . 0493 for first graph , * * P = 0 . 0015 for second graph , * P = 0 . 0391 for third graph .

O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B From animal subjects in (A), in situ hybridization (ISH, purple stain) revealed increased miR-210 in < 100-μm pulmonary vessels of mice suffering from PH, *P = 0.0493 for first graph, **P = 0.0015 for second graph, *P = 0.0391 for third graph.


C Representative ISH stain of miR - 210 in < 100 μm pulmonary vessels of mice ( bottom ) exposed to Hyp + SU5416 compared with Norm + SU5416 ( α - smooth muscle actin stain from serial sections , top row of ) .

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O

C Representative ISH stain of miR-210 in < 100 μm pulmonary vessels of mice (bottom micrographs) exposed to Hyp + SU5416 compared with Norm + SU5416 (α-smooth muscle actin stain from serial sections, top row of micrographs).


D Increased miR - 210 in < 200 - μm remodeled pulmonary vessels of suffering from PAH ( N = 19 , Supplementary Table S1 ) as compared with non - PAH donor control lung ( N = 10 ) . Serial with hematoxylin eosin is displayed in top row of ; of miRNA ISH , right graph , * P = 0 . 0167 .

O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Increased miR-210 in < 200-μm remodeled pulmonary vessels of patients suffering from PAH (N = 19, Supplementary Table S1) as compared with non-PAH donor control lung (N = 10). Serial staining with hematoxylin and eosin is displayed in the top row of micrographs; quantification of miRNA ISH, right graph, *P = 0.0167.


E Increased of miR - 210 in plasma drawn from pulmonary circulation ( pulmonary capillary wedge position , PCWP ) of with elevated mean pulmonary arterial pressures ( mean PAP ≥ 25 mmHg ) compared with control subjects ( mPAP < 25 mmHg , N = 5 / group , demographics in Supplementary Table S2 ) , * P = 0 . 0357 .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E Increased levels of miR-210 in plasma drawn from the pulmonary circulation (pulmonary capillary wedge position, PCWP) of patients with elevated mean pulmonary arterial pressures (mean PAP ≥ 25 mmHg) compared with control subjects (mPAP < 25 mmHg, N = 5/group, demographics in Supplementary Table S2), *P = 0.0357.


F From in ( C ) immunohistochemistry ( ) revealed that miR - 210 targets ISCU1 / 2 were reciprocally down - regulated in miR - 210 - enriched remodeled pulmonary vessels - namely in PH mice exposed to Hyp + SU5416 ( F , * * * P = 0 . 0002 ) .

O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F From animals in (C) immunohistochemistry (IHC) revealed that the miR-210 targets ISCU1/2 were reciprocally down-regulated in miR-210-enriched remodeled pulmonary vessels-namely in PH mice exposed to Hyp + SU5416 (F, ***P = 0.0002).


G From humans in ( D ) , immunohistochemistry ( ) revealed that miR - 210 targets ISCU1 / 2 were reciprocally down - regulated in miR - 210 - enriched remodeled pulmonary vessels - namely in human PAH ( G , * * * P = 0 . 0008 ) .

O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G From humans in (D), immunohistochemistry (IHC) revealed that the miR-210 targets ISCU1/2 were reciprocally down-regulated in miR-210-enriched remodeled pulmonary vessels-namely in human PAH patients (G, ***P = 0.0008).


H , I miR - 210 expression ( H ) was increased ( * * * P = 0 . 0003 ) , ISCU1 / 2 expression ( I ) was decreased ( * * * P < 0 . 0001 ) in PECAM + pulmonary vascular endothelial isolated from PH mice ( Hyp + SU5416 ) as compared with control ( Norm + SU5416 ) ( N = 4 / group , left bars ) .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H, I miR-210 expression (H) was increased (***P = 0.0003), and ISCU1/2 expression (I) was decreased (***P < 0.0001) in PECAM+pulmonary vascular endothelial cells isolated from PH mice (Hyp + SU5416) as compared with control (Norm + SU5416) (N = 4/group, left bars).


After lentiviral delivery of GCN4 or GRX2 sensor genes to human PAECs , was measured by flow cytometry . Unlike control GCN4 sensors that homodimerized independent of Fe - S induced consistent , derived from GRX2 Fe - S - dependent sensors decreased in hypoxia , as displayed in representative flow cytometric plots ( left ) by of of positive [ ( number in M2 gate ) / ( total number ) × 100 ] . Immunoblotting revealed consistent expression of either GRX2 or GCN4 sensors ( FLAG - tagged ) in hypoxia ( Hyp ) compared with normoxia ( Norm ) ( N = 3 , * * * P < 0 . 0001 for GRX2 ; N = 3 , NS P = 0 . 1848 for GCN4 ) . In contrast to consistent from control GCN4 sensors , Fe - S - dependent GRX2 sensor was decreased by siRNA knockdown of ISCU1 / 2 ( siISCU ) as compared with control ( siCont ) ( N = 3 , * * * P < 0 . 0001 for GRX2 ; N = 3 , NS P = 0 . 1790 for GCN4 ) .

O O O O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O

After lentiviral delivery of GCN4 or GRX2 sensor genes to human PAECs, cellular fluorescence was measured by flow cytometry. Unlike control GCN4 sensors that homodimerized independent of Fe-S levels and induced consistent fluorescence, fluorescence derived from the GRX2 Fe-S-dependent sensors decreased in hypoxia, as displayed in representative flow cytometric plots (left) and by quantification of percentage of positive cells [(cell number in M2 gate)/(total cell number) × 100]. Immunoblotting revealed consistent expression of either GRX2 or GCN4 sensors (FLAG-tagged) in hypoxia (Hyp) compared with normoxia (Norm) (N = 3, ***P < 0.0001 for GRX2; N = 3, NS P = 0.1848 for GCN4).In contrast to consistent fluorescence from control GCN4 sensors, Fe-S-dependent GRX2 sensor fluorescence was decreased by siRNA knockdown of ISCU1/2 (siISCU) as compared with control (siCont) (N = 3, ***P < 0.0001 for GRX2; N = 3, NS P = 0.1790 for GCN4).


GRX2 , but not GCN4 , sensor was decreased after transfection of miR - 210 oligonucleotide mimic ( miR - 210 ) as compared with control ( miRC ) ( N = 3 , * * * P = 0 . 0002 for GRX2 ; N = 3 , NS P = 0 . 0913 for GCN4 ) . During hypoxic exposure , GRX2 , but not GCN4 , sensor was increased after transfection of an antisense miR - 210 inhibitor ( AS210 ) as compared with control ( ASC ) ( N = 3 , * * P = 0 . 003 for GRX2 ; N = 3 , NS P = 0 . 1194 for GCN4 ) .

B-CONTROLLED_VAR O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O

GRX2, but not GCN4, sensor fluorescence was decreased after transfection of miR-210 oligonucleotide mimic (miR-210) as compared with control (miRC) (N = 3, ***P = 0.0002 for GRX2; N = 3, NS P = 0.0913 for GCN4). During hypoxic exposure, GRX2, but not GCN4, sensor fluorescence was increased after transfection of an antisense miR-210 inhibitor (AS210) as compared with control (ASC) (N = 3, **P = 0.003 for GRX2; N = 3, NS P = 0.1194 for GCN4).


After lentiviral delivery of sensor genes , Fe - S - dependent GRX2 sensor was decreased in PECAM - positive from PH mouse lung ( Hyp + SU5416 ) as compared with control non - PH mouse lung ( Norm + SU5416 , N = 3 , * * * P < 0 . 001 ) .

O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

After lentiviral delivery of sensor genes, Fe-S-dependent GRX2 sensor fluorescence was decreased in PECAM-positive cells from PH mouse lung (Hyp + SU5416) as compared with control non-PH mouse lung (Norm + SU5416, N = 3, ***P < 0.001).


By electron paramagnetic resonance ( EPR ) spectroscopy ( representative VHL − / − versus control lung ) , Fe - S cluster signal was decreased ( left graph ) in VHL − / − mice lung tissue ( VHL − / − , N = 3 ) as compared with WT control lung tissues ( WT , N = 5 ) , * * * P = 0 . 0003 .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

By electron paramagnetic resonance (EPR) spectroscopy (representative VHL−/− versus control lung), Fe-S cluster signal was decreased (left graph) in VHL−/− mice lung tissue (VHL−/−, N = 3) as compared with WT control lung tissues (WT, N = 5), ***P = 0.0003.


By EPR , Fe - S - specific signal was decreased ( right graph ) in human PH - diseased lung tissue harvested at lung transplantation ( PH , N = 3 , Supplementary Table S3 ) compared with non - PH control donor lung tissues ( control , N = 3 ) , * P = 0 . 048 .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

By EPR, Fe-S-specific signal was decreased (right graph) in human PH-diseased lung tissue harvested at lung transplantation (PH, N = 3, Supplementary Table S3) compared with non-PH control donor lung tissues (control, N = 3), *P = 0.048.


B By immunohistochemistry ( ) , ISCU1 / 2 was unchanged in < 100 μm pulmonary vessels of miR - 210 − / − mice exposed to Hyp + SU5416 ( N = 6 ) versus Norm + SU5416 ( N = 7 ) , NS P = 0 . 2833 .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B By immunohistochemistry (IHC), ISCU1/2 was unchanged in < 100 μm pulmonary vessels of miR-210−/−mice exposed to Hyp + SU5416 (N = 6) versus Norm + SU5416 (N = 7), NS P = 0.2833.


C Fe - S - dependent I - specific activity was decreased in WT PH mice as compared with control ( N = 7 / group , left bars , * * * P < 0 . 0001 ) , but activity was preserved in lungs of miR - 210 − / − mice in either condition ( N = 8 / group , right bars ) , NS P = 0 . 3693 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

C Fe-S-dependent Complex I-specific activity was decreased in WT PH mice as compared with control (N = 7/group, left bars, ***P < 0.0001), but activity was preserved in the lungs of miR-210−/−mice in either condition (N = 8/group, right bars), NS P = 0.3693.


D demonstrated that pulmonary vascular GLUT1 was increased in WT PH mice ( left bars , * * * P = 0 . 0006 ) , but GLUT1 was unchanged in miR - 210 − / − mice in either condition ( right bars , NS P = 0 . 9967 ) .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

D IHC demonstrated that pulmonary vascular GLUT1 was increased in WT PH mice (left bars, ***P = 0.0006), but GLUT1 was unchanged in miR-210−/− mice in either condition (right bars, NS P = 0.9967).


E 3 - nitrotyrosine ( 3 - NT ) was increased in pulmonary vessels of WT PH mice but was reduced in miR - 210 − / − mice in either condition ( N = 6 / group ) ( * * * P < 0 . 0001 , NS P = 0 . 4087 ) .

O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E 3-nitrotyrosine (3-NT) was increased in pulmonary vessels of WT PH mice but was reduced in miR-210−/− mice in either condition (N = 6/group) (***P < 0.0001, NS P = 0.4087).


F In PH conditions , endothelin - 1 was decreased in miR - 210 − / − lung tissue compared with WT tissue ( N = 5 / group ) , * * P = 0 . 007 .

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

F In PH conditions, endothelin-1 was decreased in miR-210−/− lung tissue compared with WT tissue (N = 5/group), **P = 0.007.


G PCNA was increased in WT PH pulmonary vessels but was decreased in miR - 210 − / − tissue exposed to either condition ( N = 5 / group ) ( * * P = 0 . 0096 , * P = 0 . 0124 , * P = 0 . 0263 for miR - 210 − / − ) .

O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O

G PCNA was increased in WT PH pulmonary vessels but was decreased in miR-210−/− tissue exposed to either condition (N = 5/group) (**P = 0.0096, *P = 0.0124, *P = 0.0263 for miR-210−/−).


H PCNA was increased in PECAM + endothelial from PH versus non - PH mice ( N = 3 / group , * * P = 0 . 0052 ) .

O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H PCNA was increased in PECAM+ endothelial cells from PH versus non-PH mice (N = 3/group, **P = 0.0052).


I Unlike WT mice ( black bars ) demonstrating increased right ventricular systolic pressure ( RVSP ) after Hyp + SU5416 ( N = 10 ) versus Norm + SU5416 ( N = 11 ) , hemodynamic dysregulation was significantly alleviated in miR - 210 − / − mice ( bars , N = 11 ) ( * * * P < 0 . 0001 , * P = 0 . 0258 , * * P = 0 . 0059 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I Unlike WT mice (black bars) demonstrating increased right ventricular systolic pressure (RVSP) after Hyp + SU5416 (N = 10) versus Norm + SU5416 (N = 11), hemodynamic dysregulation was significantly alleviated in miR-210−/− mice (white bars, N = 11) (***P < 0.0001, *P = 0.0258, **P = 0.0059).


J Compared with WT controls ( N = 9 ) , miR - 210 − / − mice ( N = 8 ) displayed a blunted increase of Fulton index ( RV / LV + S ) under PH versus baseline conditions ( expressed as a of RV / LV + S under Hyp + SU5416 versus Norm + SU5416 , * P = 0 . 031 ) .

O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

J Compared with WT controls (N = 9), miR-210−/− mice (N = 8) displayed a blunted increase of the Fulton index (RV/LV + S) under PH versus baseline conditions (expressed as a ratio of RV/LV + S under Hyp + SU5416 versus Norm + SU5416, *P = 0.031).


K - M Under PH ( black bars , N = 8 / group ) versus baseline conditions ( bars , N = 6 / group ) , pulmonary vascular remodeling was alleviated in miR - 210 − / − mice , as visualized via histology ( L ) , confirmed by decreased % arteriolar muscularization ( K , * * P = 0 . 001 , * * P = 0 . 0045 for miR - 210 − / − , * P = 0 . 0158 ) decreased vessel wall thickness ( M , * * * P < 0 . 0001 , * * P = 0 . 0086 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

K-M Under PH (black bars, N = 8/group) versus baseline conditions (white bars, N = 6/group), pulmonary vascular remodeling was alleviated in miR-210−/− mice, as visualized via histology (L), and confirmed by decreased % arteriolar muscularization (K, **P = 0.001, **P = 0.0045 for miR-210−/−, *P = 0.0158) and decreased vessel wall thickness (M, ***P < 0.0001, **P = 0.0086).


B , C Intrapharyngeal delivery of miR - 210 mimic increased miR - 210 in whole lung ( * * * P = 0 . 0002 ) ( B ) in < 100 - μm pulmonary vessels ( * * P = 0 . 007 ) ( C ) .

O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B, C Intrapharyngeal delivery of miR-210 mimic increased miR-210 in whole lung (***P = 0.0002) (B) and in < 100-μm pulmonary vessels (**P = 0.007) (C).


D miR - 210 mimic also repressed ISCU1 / 2 in those same caliber vessels ( N = 6 / group ) , * * P = 0 . 0022 .

O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O

D miR-210 mimic also repressed ISCU1/2 levels in those same caliber vessels (N = 6/group), **P = 0.0022.


E Endothelin - 1 was increased in mouse lung tissue after delivery of miR - 210 mimic ( N = 6 / group ) , * P = 0 . 0368 .

O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O

E Endothelin-1 was increased in mouse lung tissue after delivery of miR-210 mimic (N = 6/group), *P = 0.0368.


F - H miR - 210 mimic delivery increased RVSP ( N = 8 / group ) ( F ) vascular remodeling ( α - smooth muscle actin stain , α - SMA ) , as evidenced by increased of muscularized ( < 100 μm ) pulmonary vessels ( G ) increased medial thickening relative to vessel diameter when compared with miR - Control ( H ) ( N = 6 / group ) . * * P = 0 . 0021 for ( F ) , * * P = 0 . 0072 for ( G ) , * P = 0 . 0335 for ( H ) .

O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F-H miR-210 mimic delivery increased RVSP (N = 8/group) (F) and vascular remodeling (α-smooth muscle actin stain, α-SMA), as evidenced by increased percent of muscularized (< 100 μm) pulmonary vessels (G) and increased medial thickening relative to vessel diameter when compared with miR-Control (H) (N = 6/group). **P = 0.0021 for (F), **P = 0.0072 for (G), *P = 0.0335 for (H).


B - D Intravenous delivery of anti - miR - 210 down - regulated miR - 210 expression in whole lung tissue ( N = 7 / group ) , * * * P < 0 . 0001 ( B ) , pulmonary vasculature as assessed by in situ ( N = 7 / group ) , * * * P = 0 . 0003 ( C ) , PECAM + vascular endothelial derived from whole lung tissue ( N = 4 / group ) , * * P = 0 . 0013 ( D ) .

O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B-D Intravenous delivery of anti-miR-210 down-regulated miR-210 expression in whole lung tissue (N = 7/group), ***P < 0.0001 (B), the pulmonary vasculature as assessed by in situ staining (N = 7/group), ***P = 0.0003 (C), and PECAM+vascular endothelial cells derived from whole lung tissue (N = 4/group), **P = 0.0013 (D).


E Correspondingly , anti - miR - 210 delivery resulted in preservation of ISCU1 / 2 expression in < 100 - μm pulmonary vessels as compared to anti - miR - Control ( N = 5 / group ) , * * P = 0 . 0011 .

O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E Correspondingly, anti-miR-210 delivery resulted in preservation of ISCU1/2 expression in < 100-μm pulmonary vessels as compared to anti-miR-Control (N = 5/group), **P = 0.0011.


F Similarly , ISCU1 / 2 expression was preserved in PECAM + endothelial derived from mice treated with anti - miR - 210 ( N = 4 / group ) , * * P = 0 . 0016 .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O

F Similarly, ISCU1/2 expression was preserved in PECAM+ endothelial cells derived from mice treated with anti-miR-210 (N = 4/group), **P = 0.0016.


G Endothelin - 1 was decreased in lung tissue from mice treated with anti - miR - 210 during Hyp + SU5416 exposure ( N = 6 / group ) , * P = 0 . 0181 .

O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O

G Endothelin-1 was decreased in lung tissue from mice treated with anti-miR-210 during Hyp + SU5416 exposure (N = 6/group), *P = 0.0181.


H As assessed by in situ immunofluorescence , PCNA was decreased in pulmonary vessels ( < 100 μm ) after anti - miR - 210 delivery , * * P = 0 . 001 .

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O

H As assessed by in situ immunofluorescence, PCNA was decreased in pulmonary vessels (< 100 μm) after anti-miR-210 delivery, **P = 0.001.


I - K Anti - miR - 210 delivery ( N = 10 / group ) ameliorated elevation of RVSP , * * P = 0 . 0031 ( i ) vascular remodeling , as reflected by increased of muscularized arterioles ( N = 8 / group , * * P = 0 . 0091 ) ( J ) increased medial thickness relative to vessel diameter in < 100 - μm pulmonary vessels ( N = 6 / group , * * P = 0 . 0021 ) ( K ) .

O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I-K Anti-miR-210 delivery (N = 10/group) ameliorated the elevation of RVSP, **P = 0.0031 (i) and vascular remodeling, as reflected by increased percent of muscularized arterioles (N = 8/group, **P = 0.0091) (J) and increased medial thickness relative to vessel diameter in < 100-μm pulmonary vessels (N = 6/group, **P = 0.0021) (K).


M Pharmacologic inhibition of miR - 210 ( N = 7 / group ) down - regulated pulmonary expression of miR - 210 , * * * P < 0 . 0001 ( M ) .

O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O

M Pharmacologic inhibition of miR-210 (N = 7/group) down-regulated pulmonary expression of miR-210, ***P < 0.0001 (M).


N Pharmacologic inhibition of miR - 210 ( N = 7 / group ) preserved ISCU1 / 2 expression in < 100 - μm pulmonary vessels , * * P = 0 . 0015 ( N ) .

O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O

N Pharmacologic inhibition of miR-210 (N = 7/group) preserved ISCU1/2 expression in < 100-μm pulmonary vessels, **P = 0.0015 (N).


O - Q In contrast to anti - miR - Control ( N = 8 ) , anti - miR - 210 ameliorated elevation of RVSP ( N = 10 / group , * * * P < 0 . 0001 ) ( O ) lung remodeling ( N = 7 / group , * * P = 0 . 0022 , * P = 0 . 0167 ) ( P - Q ) .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

O-Q In contrast to anti-miR-Control (N = 8), anti-miR-210 ameliorated the elevation of RVSP (N = 10/group, ***P < 0.0001) (O) and lung remodeling (N = 7/group, **P = 0.0022, *P = 0.0167) (P-Q).


A Following schema of Fig5A in presence of hypoxia but absence of SU5416 , intravenous delivery of anti - miR - 210 ameliorated elevation of RVSP compared with anti - miR - Control ( N = 6 / group , * * * P = 0 . 001 ) .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Following the schema of Fig5A in the presence of hypoxia but the absence of SU5416, intravenous delivery of anti-miR-210 ameliorated the elevation of RVSP compared with anti-miR-Control (N = 6/group, ***P = 0.001).


C , D Consistent with specific delivery to vascular endothelium after intravenous administration ( Dahlman et al , 2014 ) , 7C1 - mediated delivery of anti - miR - 210 ( N = 6 / group ) decreased miR - 210 expression in PECAM + pulmonary vascular endothelial , * * P = 0 . 0013 , ( D ) but not PECAM - negative pulmonary , NS P = 0 . 3775 ( C ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C, D Consistent with specific delivery to the vascular endothelium after intravenous administration (Dahlman et al, 2014), 7C1-mediated delivery of anti-miR-210 (N = 6/group) decreased miR-210 expression in PECAM+ pulmonary vascular endothelial cells, **P = 0.0013, (D) but not PECAM-negative pulmonary cells, NS P = 0.3775 (C).


E Correspondingly , endothelial - specific anti - miR - 210 ( N = 6 / group ) increased ISCU1 / 2 expression in PECAM + even in presence of chronic hypoxia , * * * P = 0 . 0003 .

O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O

E Correspondingly, endothelial-specific anti-miR-210 (N = 6/group) increased ISCU1/2 expression in PECAM+ cells even in the presence of chronic hypoxia, ***P = 0.0003.


F - I Delivery of anti - miR - 210 ( N = 5 / group ) decreased endothelin - 1 in hypoxic mouse lung tissue , * P = 0 . 0198 ( F ) . As a result , in contrast to anti - miR - Control ( N = 8 ) , endothelial - specific anti - miR - 210 ameliorated elevation of RVSP ( N = 6 ) , * * P = 0 . 0024 ( G ) , pulmonary vascular remodeling ( N = 6 / group ) , * * P = 0 . 0021 , * P = 0 . 0117 ( H - I ) .

O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F-I Delivery of anti-miR-210 (N = 5/group) decreased endothelin-1 in hypoxic mouse lung tissue, *P = 0.0198 (F). As a result, in contrast to anti-miR-Control (N = 8), endothelial-specific anti-miR-210 ameliorated the elevation of RVSP (N = 6), **P = 0.0024 (G), and pulmonary vascular remodeling (N = 6/group), **P = 0.0021, *P = 0.0117 (H-I).


B , C Pharmacologic inhibition of ISCU ( N = 8 / group ) did not influence miR - 210 expression in lungs either after Norm + SU5416 , NS P = 0 . 8634 ( B ) or Hyp + SU5416 , NS P = 0 . 9786 ( C ) .

O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B, C Pharmacologic inhibition of ISCU (N = 8/group) did not influence miR-210 expression in the lungs either after Norm + SU5416, NS P = 0.8634 (B) or Hyp + SU5416, NS P = 0.9786 (C).


D As compared with control siRNA ( siCont ) where ISCU1 / 2 expression was decreased after exposure to Hyp + SU5416 ( bars ) , lung delivery of siRNA specific for ISCU1 / 2 ( siISCU ) via Staramine - mPEG nanocomplexes ( Polach et al , 2012 ) down - regulated ISCU1 / 2 in < 100 - μm pulmonary vessels in Norm + SU5416 Hyp + SU5416 ( N = 5 / group , * * * P = 0 . 0006 , * * P = 0 . 0019 for Hyp + SU5416 , * * P = 0 . 0029 for siCont ) .

O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D As compared with control siRNA (siCont) where ISCU1/2 expression was decreased after exposure to Hyp + SU5416 (white bars), lung delivery of siRNA specific for ISCU1/2 (siISCU) via Staramine-mPEG nanocomplexes (Polach et al, 2012) down-regulated ISCU1/2 in < 100-μm pulmonary vessels in Norm + SU5416 and Hyp + SU5416 (N = 5/group, ***P = 0.0006, **P = 0.0019 for Hyp + SU5416, **P = 0.0029 for siCont).


E By electron paramagnetic resonance ( EPR ) spectroscopy ( representative siCont versus siISCU lung ) , Fe - S cluster signal was decreased ( quantitative graph , right ) in siISCU mouselung tissue ( N = 4 mice ) compared with siCont ( N = 5 mice ) , * P = 0 . 0143 .

O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E By electron paramagnetic resonance (EPR) spectroscopy (representative siCont versus siISCU lung), Fe-S cluster signal was decreased (quantitative graph, right) in siISCU mouselung tissue (N = 4 mice) compared with siCont (N = 5 mice), *P = 0.0143.


F Endothelin - 1 was increased in lung tissue after siISCU delivery under normoxic conditions ( N = 5 / group ) , * * P = 0 . 0027 .

O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

F Endothelin-1 was increased in lung tissue after siISCU delivery under normoxic conditions (N = 5/group), **P = 0.0027.


G - I In absence ( left bars ) presence ( right bars ) of Hyp + SU5416 for 2 , siISCU ( black bars ) induced elevations in RVSP ( * * * P < 0 . 0001 , * * P = 0 . 0031 ) ( G ) increased pulmonary vascular remodeling in < 100 - μm pulmonary vessels ( H , I ) as compared with siCont ( N = 5 / group , * * * P = 0 . 0003 , * P = 0 . 0114 for H ; * * * P = 0 . 0006 , * * P = 0 . 0098 for I ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G-I In the absence (left bars) and presence (right bars) of Hyp + SU5416 for 2 weeks, siISCU (black bars) induced elevations in RVSP (***P < 0.0001, **P = 0.0031) (G) and increased pulmonary vascular remodeling in < 100-μm pulmonary vessels (H, I) as compared with siCont (N = 5/group, ***P = 0.0003, *P = 0.0114 for H; ***P = 0.0006, **P = 0.0098 for I).


G - I In absence ( left bars ) presence ( right bars ) of Hyp + SU5416 for 2 , siISCU ( black bars ) induced elevations in RVSP ( * * * P < 0 . 0001 , * * P = 0 . 0031 ) ( G ) increased lung remodeling in < 100 - μm pulmonary vessels ( H , I ) as compared with siCont ( N = 5 / group , * * * P = 0 . 0003 , * P = 0 . 0114 for H ; * * * P = 0 . 0006 , * * P = 0 . 0098 for I ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G-I In the absence (left bars) and presence (right bars) of Hyp + SU5416 for 2 weeks, siISCU (black bars) induced elevations in RVSP (***P < 0.0001, **P = 0.0031) (G) and increased lung remodeling in < 100-μm pulmonary vessels (H, I) as compared with siCont (N = 5/group, ***P = 0.0003, *P = 0.0114 for H; ***P = 0.0006, **P = 0.0098 for I).


Identification of ENU - induced hearing loss pedigree MPC169 , subsequently named clarinet . ABR phenotyping of pedigree Muta - Ped - C3PDE - 169 at 3 - months of identified 8 mice with elevated hearing thresholds ( red triangles ) compared to their normal hearing colony mates ( n = 61 , black triangles ) . Indeed , eight affected mice were found to not respond to highest stimulus ( 90 dB SPL ) at three frequencies tested , or click stimulus , so their thresholds are shown as 95 dB SPL .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Identification of the ENU-induced hearing loss pedigree MPC169, subsequently named clarinet. ABR phenotyping of pedigree Muta-Ped-C3PDE-169 at 3-months of age identified 8 mice with elevated hearing thresholds (red triangles) compared to their normal hearing colony mates (n=61, black triangles). Indeed, all eight affected mice were found to not respond to the highest intensity stimulus (90 dB SPL) at the three frequencies tested, or the click stimulus, and so their thresholds are shown as 95 dB SPL.


Averaged ABR thresholds for Clrn2clarinet / del629 compound heterozygotes at P21 , showing significantly elevated thresholds compared to Clrn2 + / + , Clrn2clarinet / + Clrn2del629 / + control colony mates . five Clrn2clarinet / del629 mice were found to not respond at highest stimulus ( 90 dB SPL ) for at least one frequency / click stimulus . Data shown are mean ± s . d . * * * p < 0 . 001 , one - way ANOVA ( Please see Appendix supplemental table 1 for exact P - values ) .

O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Averaged ABR thresholds for Clrn2clarinet/del629 compound heterozygotes at P21, showing significantly elevated thresholds compared to Clrn2+/+, Clrn2clarinet/+ and Clrn2del629/+ control colony mates. All five Clrn2clarinet/del629 mice were found to not respond at the highest intensity stimulus (90 dB SPL) for at least one frequency/click stimulus. Data shown are mean ± s.d. ***p<0.001, one-way ANOVA (Please see Appendix supplemental table 1 for exact P-values).


Regional plot of p values for SNP association with hearing difficulty around CLRN2 gene locus . genes within region are annotated , direction of transcripts is shown by arrows . Colouring is on linkage disequilibrium ( LD ) across region with most associated SNP , rs35414371 , shown in purple .

O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Regional plot of p values for SNP association with hearing difficulty around the CLRN2 gene locus. The genes within the region are annotated, and the direction of the transcripts is shown by arrows. Colouring is based on linkage disequilibrium (LD) across the region with the most associated SNP, rs35414371, shown in purple.