Under the same growth conditions ( permitted water , free food , and a 12 h / 12 h dark / light cycle at 20 ± 2 ° C ) , eight - week - old male C57BL / 6 mice were continuously fed with methionine - and choline - supplemented ( MCS ) or methionine - and choline - deficient ( MCD ) diets for 6 weeks , and at the same time , gavaged with LicoB , MCC950 n = 6 mice per group Real - time quantitative PCR was used to detect the mRNA levels of Col1a1 ( E ) , TNF - α ( F ) in the mice livers , as described in ( A ) ( n = 6 mice per 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 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 O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR 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 O O O O O O O O O O B-MEASURED_VAR 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

Under the same growth conditions (permitted water, free food, and a 12 h/12 h dark/light cycle at 20±2°C), eight-week-old male C57BL/6 mice were continuously fed with methionine- and choline-supplemented (MCS) or methionine- and choline-deficient (MCD) diets for 6 weeks, and at the same time, gavaged with LicoB, MCC950 n=6 mice per group Real-time quantitative PCR was used to detect the mRNA levels of Col1a1 (E), TNF-α (F) in the mice livers, as described in (A) (n=6 mice per group).


Under the same growth conditions ( permitted water , free food , and a 12 h / 12 h dark / light cycle at 20 ± 2 ° C ) , eight - week - old male C57BL / 6 mice were continuously fed with methionine - and choline - supplemented ( MCS ) or methionine - and choline - deficient ( MCD ) diets for 6 weeks , and at the same time , gavaged with LicoB , MCC950 n = 6 mice per group Real - time quantitative PCR was used to detect the mRNA levels of IL - 1β ( G ) , and IL - 18 ( H ) in the mice livers , as described in ( A ) ( n = 6 mice per 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 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 O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR 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 O O O O O 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 O O O O O O O O O O O O O O O O O O

Under the same growth conditions (permitted water, free food, and a 12 h/12 h dark/light cycle at 20±2°C), eight-week-old male C57BL/6 mice were continuously fed with methionine- and choline-supplemented (MCS) or methionine- and choline-deficient (MCD) diets for 6 weeks, and at the same time, gavaged with LicoB, MCC950 n=6 mice per group Real-time quantitative PCR was used to detect the mRNA levels of IL-1β (G), and IL-18 (H) in the mice livers, as described in (A) (n=6 mice per group).


A . Flow cytometry of cells isolated from heart tissue when scaled against TMRM staining and also FSC and SSC parameters of flow cytometry .

O 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. Flow cytometry of cells isolated from heart tissue when scaled against TMRM staining and also FSC and SSC parameters of flow cytometry.


B . Cardiac troponin I ( cTnI ) and α - Sarcomeric actinin ( α - SA ) staining of the TMRM + + population ( CMs ) and Vimentin staining of the TMRM + population ( NCs ) . The scale bar represents 100 µm .

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

B. Cardiac troponin I (cTnI) and α-Sarcomeric actinin (α-SA) staining of the TMRM++ population (CMs) and Vimentin staining of the TMRM+ population (NCs). The scale bar represents 100 µm.


D . EGFP expression in iPSC - like colonies derived from CMs or NCs isolated from triple transgenic mice .

D. EGFP expression in iPSC-like colonies derived from CMs or NCs isolated from triple transgenic mice.


E . EGFP expression combined with immunofluorescence staining of SSEA - 1 and DAPI of iPSC - like colonies derived from CMs isolated from triple transgenic 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

E. EGFP expression combined with immunofluorescence staining of SSEA-1 and DAPI of iPSC-like colonies derived from CMs isolated from triple transgenic mice.


F . Morphological changes of isolated CMs and NCs at different time points before AP positive colony formation at day 6 . The scale bar represents 100 µm .

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

F. Morphological changes of isolated CMs and NCs at different time points before AP positive colony formation at day 6. The scale bar represents 100 µm.


A . Gene profiling of CM and NC at D0 - D6 as shown clustered into 8 categories . Red indicates up - regulated genes while green indicates down - regulated genes ( n = 3 , n = 2 for NC - D6 ) .

O O O O O O O O O O O O O O O O O O O O O O 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. Gene profiling of CM and NC at D0-D6 as shown clustered into 8 categories. Red indicates up-regulated genes while green indicates down-regulated genes (n=3, n=2 for NC-D6).


B . Expression patterns of all genes per cluster by grey lines . Black line shows the mean value .

B. Expression patterns of all genes per cluster by grey lines. Black line shows the mean value.


D . Correlative analysis representing the relationships between CM and NC reprogramming specifically at days 2 , 4 and 6 . Red indicates closely related groups , while green indicates significantly different groups .

O O O O O O O O O O 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. Correlative analysis representing the relationships between CM and NC reprogramming specifically at days 2, 4 and 6. Red indicates closely related groups, while green indicates significantly different groups.


A . RNA expression ratio ( D2 - to - D0 ) of candidates selected from day 2 microarray data .

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

A. RNA expression ratio (D2-to-D0) of candidates selected from day 2 microarray data.


C . Ki67 , α - MHC , and DAPI staining and quantification of CMs treated with control ( Ctrl ) , or combinations of a FoxM1 up - regulation adenovirus ( F ) , Id1 up - regulation adenovirus ( I ) , Hmgb2 up - regulation adenovirus ( H ) or a small molecule Jnk3 inhibitor ( Ji ) .

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

C. Ki67, α-MHC, and DAPI staining and quantification of CMs treated with control (Ctrl), or combinations of a FoxM1 up-regulation adenovirus (F), Id1 up-regulation adenovirus (I), Hmgb2 up-regulation adenovirus (H) or a small molecule Jnk3 inhibitor (Ji).


D . Ki67 , α - MHC , and DAPI staining and quantification of CMs treated with Ctrl or combination of FI with Jnk3 - shRNAs in place of the small molecule ( Ji ) . The arrow head indicates Ki67 + / α - MHC + proliferated CMs .

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 B-CONTROLLED_VAR O O O O 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

D. Ki67, α-MHC, and DAPI staining and quantification of CMs treated with Ctrl or combination of FI with Jnk3-shRNAs in place of the small molecule (Ji). The arrow head indicates Ki67+/α-MHC+ proliferated CMs.


E . Morphology and quantification of FIJs - treated CMs undergoing cytokinesis by H3P and Aurora kinase B ( AURKB ) staining .

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

E. Morphology and quantification of FIJs-treated CMs undergoing cytokinesis by H3P and Aurora kinase B (AURKB) staining.


G . RNA expression ratio of Oct4 , FoxM1 , Id1 , or Jnk3 in doxycycline - injected versus control adult CMs isolated from reprogrammable mice .

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

G. RNA expression ratio of Oct4, FoxM1, Id1, or Jnk3 in doxycycline-injected versus control adult CMs isolated from reprogrammable mice.


H . Immunofluorescence of H3P and cTnI and quantification of the heart tissue sections from control or doxycycline injected mice .

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

H. Immunofluorescence of H3P and cTnI and quantification of the heart tissue sections from control or doxycycline injected mice.


B . The size of hearts isolated from Ctrl or FIJs - treated mice ; heart / body weight ratio of Ctrl or FIJs - treated mice .

B. The size of hearts isolated from Ctrl or FIJs-treated mice; heart/body weight ratio of Ctrl or FIJs-treated mice.


C . H3P , cTnI , and DAPI staining and quantification of heart tissues in Ctrl or FIJs - treated mice . The arrowhead indicates H3P + / cTnI + proliferated CMs and the scale bar represents 50 µm .

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

C. H3P, cTnI, and DAPI staining and quantification of heart tissues in Ctrl or FIJs-treated mice. The arrowhead indicates H3P+/cTnI+ proliferated CMs and the scale bar represents 50 µm.


E . Immunofluorescence of H3P and cTnI and quantification of the heart tissue sections from adeno - Ctrl or adeno - FIJs injected mice .

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

E. Immunofluorescence of H3P and cTnI and quantification of the heart tissue sections from adeno-Ctrl or adeno-FIJs injected mice.


F . Quantification of BrdU + / EGFP + population % in isolated CMs from adeno - Ctrl or adeno - FIJs injected M / Z mice by flow cytometry .

O O O O O O O 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. Quantification of BrdU+/EGFP+ population % in isolated CMs from adeno-Ctrl or adeno-FIJs injected M/Z mice by flow cytometry.


B . RNA expressional changes of FoxM1 , Id1 , and Jnk3 after adeno - Ctrl or adeno - FIJs treatment after MI .

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

B. RNA expressional changes of FoxM1, Id1, and Jnk3 after adeno-Ctrl or adeno-FIJs treatment after MI.


C . Immunofluorescence staining of BrdU or H3P and cTnI or EGFP and quantification in the heart tissues after MI . The arrow head indicates cTnI + / BrdU + , cTnI + / H3P + or EGFP + / BrdU + , EGFP + / H3P + proliferated CMs . The scale bar represents 50 µm .

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

C. Immunofluorescence staining of BrdU or H3P and cTnI or EGFP and quantification in the heart tissues after MI. The arrow head indicates cTnI+/BrdU+, cTnI+/H3P+ or EGFP+/BrdU+, EGFP+/H3P+proliferated CMs. The scale bar represents 50 µm.


D . Ejection fraction and fraction shortening percentage in Ctrl or FIJs - treated mice 21 days post - MI .

D. Ejection fraction and fraction shortening percentage in Ctrl or FIJs-treated mice 21 days post-MI.


E . Catheterization in Ctrl or FIJs - treated mice 21 days post - MI under baseline or inferior vena cava occlusion condition .

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. Catheterization in Ctrl or FIJs-treated mice 21 days post-MI under baseline or inferior vena cava occlusion condition.


F . Trichrome staining and quantification showing percentage of fibrotic tissue ( blue ) in Ctrl or FIJs - treated mice 21 days after MI .

O O 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. Trichrome staining and quantification showing percentage of fibrotic tissue (blue) in Ctrl or FIJs-treated mice 21 days after MI.


A . RNA expression of mitosis checkpoint genes in CMs with FoxM1 or Id1 overexpression , Jnk3 - shRNA , or combined treatment .

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

A. RNA expression of mitosis checkpoint genes in CMs with FoxM1 or Id1 overexpression, Jnk3-shRNA, or combined treatment.


B . Transcriptional expression of cyclin - dependent kinases in FoxM1 or Id1 up - regulated , Jnk3 down - regulated , or combined treated CMs .

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

B. Transcriptional expression of cyclin-dependent kinases in FoxM1 or Id1 up-regulated, Jnk3 down-regulated, or combined treated CMs.


C . Real - time RT - PCR analysis of CDK inhibitor expression in CMs with FoxM1 or Id1 overexpression , Jnk3 - shRNA or combined treatment .

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 B-CONTROLLED_VAR O O O O O O

C. Real-time RT-PCR analysis of CDK inhibitor expression in CMs with FoxM1 or Id1 overexpression, Jnk3-shRNA or combined treatment.


a , Starvation ( 3 h ) - induced autophagy results in a downregulation of H4K16ac in histone extracts of MEF cells .

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

a, Starvation (3 h)-induced autophagy results in a downregulation of H4K16ac in histone extracts of MEF cells.


b , Upon rapamycin treatment ( 300 nM ) LC3 conversion and downregulation of H4K16ac are observed in WT MEF cells but not in the autophagy - deficient atg5 − / − and atg7 − / − MEF cells .

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

b, Upon rapamycin treatment (300 nM) LC3 conversion and downregulation of H4K16ac are observed in WT MEF cells but not in the autophagy-deficient atg5−/− and atg7−/− MEF cells.


c , Rapamycin treatment increased the LC3 - II / LC3 - I ratio and promoted H4K16ac decrease in sirt1 − / − and WT MEF cells .

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

c, Rapamycin treatment increased the LC3-II/LC3-I ratio and promoted H4K16ac decrease in sirt1−/− and WT MEF cells.


d , Rapamycin - induced autophagy led to downregulation of H4K16ac at 48 h in histone extracts of HeLa and U20S cells , and after 6 h in U1810 cells .

O O B-CONTROLLED_VAR 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

d, Rapamycin-induced autophagy led to downregulation of H4K16ac at 48 h in histone extracts of HeLa and U20S cells, and after 6 h in U1810 cells.


e , Quantification of H4K16 acetylation by immunoblotting is depicted for rapamycin - treated cells . Data are expressed as mean ± s . e . m . ( n = 3 - 5 ) ; * P 0 . 05 ; * * P 0 . 01 .

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

e, Quantification of H4K16 acetylation by immunoblotting is depicted for rapamycin-treated cells. Data are expressed as mean ± s.e.m. (n = 3-5); *P 0.05; **P 0.01.


Rapamycin treatment ( 48 h ) promoted the downregulation of the H4K16 histone acetyltransferase hMOF expression level in MEF ( a )

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

Rapamycin treatment (48 h) promoted the downregulation of the H4K16 histone acetyltransferase hMOF expression level in MEF (a)


Rapamycin treatment ( 48 h ) promoted the downregulation of the H4K16 histone acetyltransferase hMOF expression level in transfected HeLa cells ( b ) .

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

Rapamycin treatment (48 h) promoted the downregulation of the H4K16 histone acetyltransferase hMOF expression level in transfected HeLa cells (b).


VPA ( 1 mM ) treatment counteracted rapamycin - induced H4K16ac downregulation ( c ) and decreased the LC3 ratio ( d ) .

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

VPA (1 mM) treatment counteracted rapamycin-induced H4K16ac downregulation (c) and decreased the LC3 ratio (d).


e , Co - treatment with chloroquine ( CQ , 10 µM ) showed that the decrease in LC3 ratio was a result of an increase in autophagic flux .

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

e, Co-treatment with chloroquine (CQ, 10 µM) showed that the decrease in LC3 ratio was a result of an increase in autophagic flux.


f , Inhibition of autophagy by CQ after hMOF overexpression shows that hMOF does not inhibit autophagic flux .

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

f, Inhibition of autophagy by CQ after hMOF overexpression shows that hMOF does not inhibit autophagic flux.


g , The yeast homologue of hMOF , Sas2 , tagged with 3 × HA showed a complete disappearance of HA signal upon autophagy induction after 3 h .

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

g, The yeast homologue of hMOF, Sas2, tagged with 3× HA showed a complete disappearance of HA signal upon autophagy induction after 3 h.


h , Overexpression of Sas2 repressed the downregulation of H4K16ac upon rapamycin treatment .

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

h, Overexpression of Sas2 repressed the downregulation of H4K16ac upon rapamycin treatment.


a , VPA increases the autophagic flux in rapamycin - treated HeLa cells transfected with the mRFP - GFP - LC3 tandem reporter construct which allows distinction between autophagosomes ( GFP + / RFP + yellow puncta ) and autolysosomes ( GFP − / RFP + red puncta ) .

O O B-CONTROLLED_VAR 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

a, VPA increases the autophagic flux in rapamycin-treated HeLa cells transfected with the mRFP-GFP-LC3 tandem reporter construct which allows distinction between autophagosomes (GFP+/RFP+ yellow puncta) and autolysosomes (GFP−/RFP+ red puncta).


b , Confocal microscopy image of a cell treated with rapamycin and VPA depicting a high ratio of red to green LC3 puncta indicating an increase in autophagic flux .

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

b, Confocal microscopy image of a cell treated with rapamycin and VPA depicting a high ratio of red to green LC3 puncta indicating an increase in autophagic flux.


c , Co - treatment with VPA and rapamycin led to increased cell death .

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

c, Co-treatment with VPA and rapamycin led to increased cell death.


d , Co - treatment with CQ abrogated VPA + rapamycin - induced cell death .

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

d, Co-treatment with CQ abrogated VPA + rapamycin-induced cell death.


e , f , Increasing H4K16ac levels by either overexpression of hMOF , inhibition of SIRT1 by siRNA or the chemical inhibitor Ex527 promoted cell death upon rapamycin treatment .

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

e, f, Increasing H4K16ac levels by either overexpression of hMOF, inhibition of SIRT1 by siRNA or the chemical inhibitor Ex527 promoted cell death upon rapamycin treatment.


Co - treatment with the autophagy inhibitor bafilomycin A1 ( BafA , 40 nM ) ( g ) or 3 - methyladenine ( 3MA , 5 mM ) ( h ) abrogated VPA + rapamycin - induced cell death .

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

Co-treatment with the autophagy inhibitor bafilomycin A1 (BafA, 40 nM) (g) or 3-methyladenine (3MA, 5 mM) (h) abrogated VPA + rapamycin-induced cell death.


Treatment of HeLa cells upon amino - acid starvation with VPA induced cell death ( i ) , which was rescued when cells were co - treated with CQ ( j ) . Data are expressed as mean ± s . e . m . ( n = 3 ) .

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

Treatment of HeLa cells upon amino-acid starvation with VPA induced cell death (i), which was rescued when cells were co-treated with CQ (j). Data are expressed as mean ± s.e.m. (n = 3).


Male mice , housed at 30 ° C or 6 ° C for 10 days , were intraperitoneally ( IP ) administered with [ U - 13C ] glucose ( 2 g / kg ) . 15 minutes after injection , BAT was harvested for metabolic enrichment assay . ( A ) m + 6 glucose enrichment in serum and BAT . ( B ) relative glucose abundance in serum and BAT .

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

Male mice, housed at 30°C or 6°C for 10 days, were intraperitoneally (IP) administered with [U-13C]glucose (2 g/kg). 15 minutes after injection, BAT was harvested for metabolic enrichment assay. (A) m+6 glucose enrichment in serum and BAT. (B) relative glucose abundance in serum and BAT.


( D , E ) Metabolic 13C enrichments in BAT of male mice are shown as m + 3 glycolytic intermediates ( D ) , m + 2 TCA cycle intermediates ( E ) .

O O O O O O O O O O O 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,E) Metabolic 13C enrichments in BAT of male mice are shown as m+3 glycolytic intermediates (D), m+2 TCA cycle intermediates (E).


( A ) Representative immunofluorescence staining shows UCP1 ( green ) , perilipin ( red ) and DAPI ( blue ) in sWAT of mice housed at 6 ° C .

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 O O O O O O O O

(A) Representative immunofluorescence staining shows UCP1 (green), perilipin (red) and DAPI (blue) in sWAT of mice housed at 6°C.


( B - G ) Male mice , housed at 30 ° C or 6 ° C for 10 days , were administered with [ U - 13C ] glucose ( 2 g / kg , IP ) . 15 minutes after injection , sWAT and gWAT were harvested for metabolic enrichment assay . Metabolic 13C enrichments in sWAT of male mice are shown as m + 6 glucose ( B ) , m + 3 glycolytic intermediates ( C ) , and m + 2 TCA cycle intermediates ( D ) . Metabolic 13C enrichments in gWAT of male mice are shown as m + 6 glucose ( E ) , m + 3 glycolysis intermediates ( F ) , and m + 2 TCA cycle intermediates ( G ) .

O O O O O 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 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 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

(B-G) Male mice, housed at 30°C or 6°C for 10 days, were administered with [U-13C]glucose (2 g/kg, IP). 15 minutes after injection, sWAT and gWAT were harvested for metabolic enrichment assay. Metabolic 13C enrichments in sWAT of male mice are shown as m+6 glucose (B), m+3 glycolytic intermediates (C), and m+2 TCA cycle intermediates (D). Metabolic 13C enrichments in gWAT of male mice are shown as m+6 glucose (E), m+3 glycolysis intermediates (F), and m+2 TCA cycle intermediates (G).


( H ) Plasma 13CO2 enrichment , after normalizing to the [ U - 13C ] glucose tracer enrichment .

(H) Plasma 13CO2 enrichment, after normalizing to the [U-13C]glucose tracer enrichment.


Mouse brown adipocytes were differentiated from the freshly isolated cells in the stromal vascular fractions ( SVF ) of the interscapular BAT . ( A ) Upper : Oil red staining of the cells during differentiation . Lower : Relative mRNA levels of brown adipocyte markers were measured by 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 O O O O O O O O O O O O O O O O O O

Mouse brown adipocytes were differentiated from the freshly isolated cells in the stromal vascular fractions (SVF) of the interscapular BAT. (A) Upper: Oil red staining of the cells during differentiation. Lower: Relative mRNA levels of brown adipocyte markers were measured by qPCR.


Mouse brown adipocytes were differentiated from the freshly isolated cells in the stromal vascular fractions ( SVF ) of the interscapular BAT . ( B ) Glycolytic intermediates were analyzed by GC / MS , after cultured with medium containing 10 mM [ U - 13C ] glucose for 2 hours .

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

Mouse brown adipocytes were differentiated from the freshly isolated cells in the stromal vascular fractions (SVF) of the interscapular BAT. (B) Glycolytic intermediates were analyzed by GC/MS, after cultured with medium containing 10 mM [U-13C]glucose for 2 hours.


Mouse brown adipocytes were differentiated from the freshly isolated cells in the stromal vascular fractions ( SVF ) of the interscapular BAT . ( C ) TCA cycle intermediates were analyzed by GC / MS , after cultured with medium containing 10 mM [ U - 13C ] glucose for 2 hours .

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

Mouse brown adipocytes were differentiated from the freshly isolated cells in the stromal vascular fractions (SVF) of the interscapular BAT. (C) TCA cycle intermediates were analyzed by GC/MS, after cultured with medium containing 10 mM [U-13C]glucose for 2 hours.


( A ) Fully differentiated brown adipocytes were treated with β3 - AR agonist CL316 , 243 ( 10 µM ) . Relative mRNA levels of thermogenic markers were measured by qPCR .

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

(A) Fully differentiated brown adipocytes were treated with β3-AR agonist CL316,243 (10 µM). Relative mRNA levels of thermogenic markers were measured by qPCR.


( B ) Pyruvate and lactate enrichment were analyzed by GC / MS , after cultured with medium containing 10 mM [ U - 13C ] glucose and 10 µM CL316 , 243 for 2 hours .

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

(B) Pyruvate and lactate enrichment were analyzed by GC/MS, after cultured with medium containing 10 mM [U-13C]glucose and 10 µM CL316,243 for 2 hours.


( C ) Citrate and malate enrichment were analyzed by GC / MS , after cultured with medium containing 10 mM [ U - 13C ] glucose and 10 µM CL316 , 243 for 2 hours .

(C) Citrate and malate enrichment were analyzed by GC/MS, after cultured with medium containing 10 mM [U-13C]glucose and 10 µM CL316,243 for 2 hours.


( A ) oxygen consumption rate ( OCR ) of mouse brown adipocytes treated with MPC inhibitor CHC ( 2 mM ) or UK5099 ( 2 µM ) , n = 6 - 7 biological repeats .

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

(A) oxygen consumption rate (OCR) of mouse brown adipocytes treated with MPC inhibitor CHC (2 mM) or UK5099 (2 µM), n=6-7 biological repeats.


( B ) Differentiated mouse brown adipocytes were pre - treated with β3 - AR agonist CL316 , 243 for 4 hours , before cultured with medium containing 10 mM [ U - 13C ] glucose for 2 hours with CHC or UK5099 . Metabolic 13C enrichments in brown adipocytes were shown as m + 3 glycolytic intermediates , m + 2 and m + 3 TCA cycle intermediates , n = 3 biological replicates .

O O O O O O O O O O O O O O O O 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 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) Differentiated mouse brown adipocytes were pre-treated with β3-AR agonist CL316,243 for 4 hours, before cultured with medium containing 10 mM [U-13C]glucose for 2 hours with CHC or UK5099. Metabolic 13C enrichments in brown adipocytes were shown as m+3 glycolytic intermediates, m+2 and m+3 TCA cycle intermediates, n=3 biological replicates.


( C ) After housed at 6 ° C for 10 days , mice were IP injected with PBS or CHC ( 500 mg / kg ) . 30 minutes after CHC treatment , mice were IP administered with [ U - 13C ] glucose ( 2 g / kg ) . Metabolic 13C enrichments in BAT of male mice are shown as m + 3 glycolytic intermediates , m + 2 and m + 3 TCA cycle intermediates , n = 7 .

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 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

(C) After housed at 6°C for 10 days, mice were IP injected with PBS or CHC (500 mg/kg). 30 minutes after CHC treatment, mice were IP administered with [U-13C]glucose (2 g/kg). Metabolic 13C enrichments in BAT of male mice are shown as m+3 glycolytic intermediates, m+2 and m+3 TCA cycle intermediates, n=7.


( D ) Body temperature of the CHC ( left : 400 mg / kg , n = 5 ) and ( right : 500 mg / kg , n = 7 ) treated mice .

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

(D) Body temperature of the CHC (left: 400 mg/kg, n=5) and (right: 500 mg/kg, n=7) treated mice.


A , Unsupervised cluster analysis of patients with cutaneous malignant melanoma based on global gene expression ( RNA - seq ) in metastases ( n = 367 ) . Partitioning around medoids clustering algorithm was used ( k = 2 ) .

O O O O O O O O O O O O O O O O O O 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, Unsupervised cluster analysis of patients with cutaneous malignant melanoma based on global gene expression (RNA-seq) in metastases (n=367). Partitioning around medoids clustering algorithm was used (k=2).


B , Heat - map displaying the expression of bromodomain genes in metastases from patients with cutaneous malignant melanoma ( n = 367 ) . BRD4 , SMARCA4 , TRIM28 and BRPF1 are highlighted by a black bar . Gene expression is represented by z - score .

O 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-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

B, Heat-map displaying the expression of bromodomain genes in metastases from patients with cutaneous malignant melanoma (n=367). BRD4, SMARCA4, TRIM28 and BRPF1 are highlighted by a black bar. Gene expression is represented by z-score.


C , Kaplan - Meier analysis of overall survival of patients with stage III melanoma in cluster C1 ( median survival 79 . 5 months ) and C2 ( median survival 25 . 9 months ) , and of patients with stage III melanoma with high ( median survival 61 . 5 months ) or low ( median survival 107 months ) TRIM28 expression . The log - rank test was used for statistical testing of survival data .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Kaplan-Meier analysis of overall survival of patients with stage III melanoma in cluster C1 (median survival 79.5 months) and C2 (median survival 25.9 months), and of patients with stage III melanoma with high (median survival 61.5 months) or low (median survival 107 months) TRIM28 expression. The log-rank test was used for statistical testing of survival data.


C , GSEA plot of RAS signature genes in in TRIM28high and TRIM28low metastatic tumors ( n = 367 ) , and in A375 cells transduced with shT28 - 1 , shT28 - 2 or shNTC ( shLUC and shSCR ) ( n = 3 per construct ) .

O O O O O O O O O O O O O O O O O O O O O O O O O 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, GSEA plot of RAS signature genes in in TRIM28high and TRIM28low metastatic tumors (n=367), and in A375 cells transduced with shT28-1, shT28-2 or shNTC (shLUC and shSCR) (n=3 per construct).


C , Metagene profiles after CUT & RUN sequencing of TRIM28 in untransduced A375 cells and of RNAPII in A375 cells transduced with shT28 - 1 or shSCR lentivirus . The lower panel is focused on the TRIM28 metagene profile to clearer depict its binding profile around TSS .

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

C, Metagene profiles after CUT&RUN sequencing of TRIM28 in untransduced A375 cells and of RNAPII in A375 cells transduced with shT28-1 or shSCR lentivirus. The lower panel is focused on the TRIM28 metagene profile to clearer depict its binding profile around TSS.


E , TRIM28 and RNAPII occupancy at EGR1 and JUNB in A375 as determined by CUT & RUN sequencing ( PI = length - normalized pause index ) .

O O B-MEASURED_VAR 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

E, TRIM28 and RNAPII occupancy at EGR1 and JUNB in A375 as determined by CUT&RUN sequencing (PI = length-normalized pause index).


F , Immunoblots against TRIM28 and JUNB in A375 , A2058 and SK - MEL - 28 cells transduced with shSCR , shT28 - 1 or shT28 - 2 lentivirus . Results are expressed as mean ± SEM from three biological replicates ( n = 3 ) . G , Densitometry of JUNB and TRIM28 protein levels ( relative to β - actin ) in A375 , A2058 and SK - MEL - 28 melanoma cells based on the immunoblots in F . One - way ANOVA with Dunnett ' s post hoc test was used for statistical testing .

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 O 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-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

F, Immunoblots against TRIM28 and JUNB in A375, A2058 and SK-MEL-28 cells transduced with shSCR, shT28-1 or shT28-2 lentivirus. Results are expressed as mean ±SEM from three biological replicates (n=3). G, Densitometry of JUNB and TRIM28 protein levels (relative to β-actin) in A375, A2058 and SK-MEL-28 melanoma cells based on the immunoblots in F. One-way ANOVA with Dunnett's post hoc test was used for statistical testing.


A , Immunoblotting to determine the overexpression of JUNB in A375 cells transduced with pBABE - JUNB .

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

A, Immunoblotting to determine the overexpression of JUNB in A375 cells transduced with pBABE-JUNB.


B , Volcano plot displaying differentially expressed genes after RNA - seq analysis of JUNB overexpressing A375 cells ( pBABE - JUNB ) and control A375 cells transduced with empty pBABE vector ( EV ) ( n = 4 ) . Genes induced after JUNB overexpression are highlighted in yellow , and gene suppressed after JUNB overexpression are highlighted in blue .

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

B, Volcano plot displaying differentially expressed genes after RNA-seq analysis of JUNB overexpressing A375 cells (pBABE-JUNB) and control A375 cells transduced with empty pBABE vector (EV) (n=4). Genes induced after JUNB overexpression are highlighted in yellow, and gene suppressed after JUNB overexpression are highlighted in blue.


D , Heatmap displaying the induction of CXCL1 and CXCL8 , and suppression of YAP1 target genes , in A375 cells overexpressing JUNB .

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 B-CONTROLLED_VAR O

D, Heatmap displaying the induction of CXCL1 and CXCL8, and suppression of YAP1 target genes, in A375 cells overexpressing JUNB.


E , Expression of YAP1 signature genes in A375 cells after transduction with dCas9 - KRAB lentiviruses expressing gRNA targeting JUNB ( gJUNB - 1 or gJUNB - 2 ) or control gRNA ( gEGFP ) . qRT - PCR was used to determine expression levels , and results are expressed as mean ± SEM from three biological replicates ( n = 3 ) .

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

E, Expression of YAP1 signature genes in A375 cells after transduction with dCas9-KRAB lentiviruses expressing gRNA targeting JUNB (gJUNB-1 or gJUNB-2) or control gRNA (gEGFP). qRT-PCR was used to determine expression levels, and results are expressed as mean ±SEM from three biological replicates (n=3).


F , Quantification of Matrigel invasion experiments with A375 cells transduced with pBABE - JUNB ( JUNB ) or pBABE empty vector ( EV ) retrovirus . Results are expressed as mean ± SEM from three biological replicates ( n = 3 ) and the two - sided unpaired t - test was used . G , Representative images from Matrigel invasion assays in ( F ) . Scale bar is 60 μm .

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 O O O O O O O O O O O O O O O O O O O O O O O O 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, Quantification of Matrigel invasion experiments with A375 cells transduced with pBABE-JUNB (JUNB) or pBABE empty vector (EV) retrovirus. Results are expressed as mean ±SEM from three biological replicates (n=3) and the two-sided unpaired t-test was used. G, Representative images from Matrigel invasion assays in (F). Scale bar is 60 μm.


H , Quantification of Matrigel invasion experiments with A375 cells transduced with dCas9 - KRAB lentiviruses expressing gRNA targeting JUNB ( gJUNB - 1 ) or control gRNA ( gEGFP ) . Results are expressed as mean ± SEM from three biological replicates ( n = 3 ) and the two - sided unpaired t - test was used . I , Representative images from Matrigel invasion assays in ( H ) . Scale bar is 60 μm .

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

H, Quantification of Matrigel invasion experiments with A375 cells transduced with dCas9-KRAB lentiviruses expressing gRNA targeting JUNB (gJUNB-1) or control gRNA (gEGFP). Results are expressed as mean ±SEM from three biological replicates (n=3) and the two-sided unpaired t-test was used. I, Representative images from Matrigel invasion assays in (H). Scale bar is 60 μm.


J , Quantification of Matrigel invasion with A375 cells transduced with dCas9 - KRAB lentiviruses expressing gRNA targeting JUNB ( gJUNB - 1 ) or a control gRNA ( gEGFP ) , and then transfected with non - targeting siRNA ( siNTC ) or siRNA targeting YAP1 ( siYAP1 ) and TAZ ( siTAZ ) . Results are expressed as mean ± SEM from three biological replicates ( n = 3 ) . One - way ANOVA and the Tukey post hoc test were used for statistical testing .

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 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

J, Quantification of Matrigel invasion with A375 cells transduced with dCas9-KRAB lentiviruses expressing gRNA targeting JUNB (gJUNB-1) or a control gRNA (gEGFP), and then transfected with non-targeting siRNA (siNTC) or siRNA targeting YAP1 (siYAP1) and TAZ (siTAZ). Results are expressed as mean ±SEM from three biological replicates (n=3). One-way ANOVA and the Tukey post hoc test were used for statistical testing.


K , Tumor growth after subcutaneous injection of 2 . 0x106 A375 cells transduced with pBABE - JUNB ( JUNB ) or pBABE empty vector ( EV ) retrovirus ( n = 10 mice per group ) . Results are expressed as mean ± SEM . Repeated - measures ANOVA was used for the statistical test of tumor growth . L , Tumor weight after the subcutaneous injection of A375 cells as shown in ( K ) . Tumor weight was analyzed 20 days after subcutaneous injection . Results are expressed as mean ± SEM . The two - sided Mann - Whitney U - test was used for the statistical test of tumor weight .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Tumor growth after subcutaneous injection of 2.0x106 A375 cells transduced with pBABE-JUNB (JUNB) or pBABE empty vector (EV) retrovirus (n=10 mice per group). Results are expressed as mean ±SEM. Repeated-measures ANOVA was used for the statistical test of tumor growth. L, Tumor weight after the subcutaneous injection of A375 cells as shown in (K). Tumor weight was analyzed 20 days after subcutaneous injection. Results are expressed as mean ±SEM. The two-sided Mann-Whitney U-test was used for the statistical test of tumor weight.


( B ) Identification of wild - type ( WT ) , heterozygous ( HZ ) , and knockout ( KO ) mice by PCR and agarose gel electrophoresis .

O O O O O O 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) Identification of wild-type (WT), heterozygous (HZ), and knockout (KO) mice by PCR and agarose gel electrophoresis.


( C ) Immunoblotting ( IB ) of ENKD1 and α - tubulin in wild - type and Enkd1 knockout mice .

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

(C) Immunoblotting (IB) of ENKD1 and α-tubulin in wild-type and Enkd1 knockout mice.


( D - F ) Immunofluorescence images ( D ) and quantification of the density of cilia ( E , n = 20 fields from three mice ) and ciliary length ( F , n = 100 cilia from three mice ) in mouse retinas stained with antibodies against acetylated α - tubulin ( Ace - α - tubulin ) and γ - tubulin and DAPI . Scale bar , 2 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 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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O

(D-F) Immunofluorescence images (D) and quantification of the density of cilia (E, n = 20 fields from three mice) and ciliary length (F, n = 100 cilia from three mice) in mouse retinas stained with antibodies against acetylated α-tubulin (Ace-α-tubulin) and γ-tubulin and DAPI. Scale bar, 2 µm.


( G - H ) ERG recording images ( G ) and a - b wave amplitudes ( H , n = 5 mice ) show the difference in the ERG a - b wave amplitude between wild - type and Enkd1 knockout 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 O O O O O O O O O O B-CONTROLLED_VAR O O O

(G-H) ERG recording images (G) and a-b wave amplitudes (H, n = 5 mice) show the difference in the ERG a-b wave amplitude between wild-type and Enkd1 knockout mice.


( I - J ) VEP recording images ( I ) and N2 - P2 wave amplitudes ( J , n = 5 mice ) show the difference in the VEP N2 - P2 wave amplitude between wild - type and Enkd1 knockout mice .

(I-J) VEP recording images (I) and N2-P2 wave amplitudes (J, n = 5 mice) show the difference in the VEP N2-P2 wave amplitude between wild-type and Enkd1 knockout mice.


( K - M ) Immunofluorescence images ( K ) and quantification of the percentage of ciliated cells ( L , n = 3 mice ) and ciliary length ( M , n = 100 cilia from three mice ) in mouse kidneys stained with the antibody against acetylated α - tubulin and DAPI . To quantify the percentage of ciliated cells ( L ) , > 200 cells from 12 images were analyzed for each mouse . Scale bar , 3 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 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

(K-M) Immunofluorescence images (K) and quantification of the percentage of ciliated cells (L, n = 3 mice) and ciliary length (M, n = 100 cilia from three mice) in mouse kidneys stained with the antibody against acetylated α-tubulin and DAPI. To quantify the percentage of ciliated cells (L), >200 cells from 12 images were analyzed for each mouse. Scale bar, 3 µm.


( N - Q ) Images ( N ) and quantification of the percentage of flagellated sperm ( O , n = 9 mice ) , flagellar length ( P , n = 100 sperm from nine mice ) , and the percentage of sperm with normal motility among flagellated sperm ( Q , n = 9 mice ) for wild - type and Enkd1 knockout mice . To quantify the percentage of flagellated sperm ( O ) , > 200 sperm were analyzed for each mouse . To quantify the percentage of sperm with normal motility among flagellated sperm ( Q ) , > 200 flagellated sperm were analyzed for each mouse . In panel N , the arrows indicate two spermatozoa without tails . Scale bar , 50 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 O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O

(N-Q) Images (N) and quantification of the percentage of flagellated sperm (O, n = 9 mice), flagellar length (P, n = 100 sperm from nine mice), and the percentage of sperm with normal motility among flagellated sperm (Q, n = 9 mice) for wild-type and Enkd1 knockout mice. To quantify the percentage of flagellated sperm (O), >200 sperm were analyzed for each mouse. To quantify the percentage of sperm with normal motility among flagellated sperm (Q), >200 flagellated sperm were analyzed for each mouse. In panel N, the arrows indicate two spermatozoa without tails. Scale bar, 50 µm.


( R , S ) Scanning electron microscopy images of cilia ( R ) and quantification of the percentage of ciliated cells ( S , n = 9 mice ) in the mouse tracheal epithelium . To quantify the percentage of ciliated cells ( S ) , > 200 cells from six fields were analyzed for each mouse . 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(R, S) Scanning electron microscopy images of cilia (R) and quantification of the percentage of ciliated cells (S, n = 9 mice) in the mouse tracheal epithelium. To quantify the percentage of ciliated cells (S), >200 cells from six fields were analyzed for each mouse. Scale bar, 5 µm.


( A ) Immunoblot analysis of ENKD1 and GAPDH in MEFs cultured in serum - free medium for 48 h .

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

(A) Immunoblot analysis of ENKD1 and GAPDH in MEFs cultured in serum-free medium for 48 h.


( B - D ) Immunofluorescence images ( B ) and quantification of the percentage of ciliated cells ( C , n = 3 independent experiments ) and ciliary length ( D , n = 70 cilia from three independent experiments ) for MEFs cultured in serum - free medium and stained with antibodies against acetylated α - tubulin and γ - tubulin and DAPI . To quantify the percentage of ciliated cells ( C ) , > 100 cells were analyzed for each experiment . 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 O O O O O O 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 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

(B-D) Immunofluorescence images (B) and quantification of the percentage of ciliated cells (C, n = 3 independent experiments) and ciliary length (D, n = 70 cilia from three independent experiments) for MEFs cultured in serum-free medium and stained with antibodies against acetylated α-tubulin and γ-tubulin and DAPI. To quantify the percentage of ciliated cells (C), >100 cells were analyzed for each experiment. Scale bar, 5 µm.


( E ) Immunoblot analysis of ENKD1 and β - actin in RPE1 cells transfected with control or ENKD1 siRNAs and cultured in serum - free medium for 48 h .

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

(E) Immunoblot analysis of ENKD1 and β-actin in RPE1 cells transfected with control or ENKD1 siRNAs and cultured in serum-free medium for 48 h.


Immunofluorescence images ( F ) and quantification of the percentage of ciliated cells ( G , n = 3 independent experiments ) for RPE1 cells transfected with control or ENKD1 siRNAs , cultured in serum - free medium , and stained with antibodies against acetylated α - tubulin and γ - tubulin and DAPI . To quantify the percentage of ciliated cells ( G ) , > 150 cells were analyzed for each experiment . 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 O O O B-CONTROLLED_VAR 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 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

Immunofluorescence images (F) and quantification of the percentage of ciliated cells (G, n = 3 independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs, cultured in serum-free medium, and stained with antibodies against acetylated α-tubulin and γ-tubulin and DAPI. To quantify the percentage of ciliated cells (G), >150 cells were analyzed for each experiment. Scale bar, 5 µm.


quantification of ciliary length ( H , n = 100 cilia from three independent experiments ) for RPE1 cells transfected with control or ENKD1 siRNAs , cultured in serum - free

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

quantification of ciliary length (H, n = 100 cilia from three independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs, cultured in serum-free medium


( I , J ) Immunofluorescence images ( I ) and quantification of the percentage of ciliated cells ( J , n = 3 independent experiments ) for RPE1 cells transfected with control or ENKD1 siRNAs , cultured in serum - free medium , and stained with antibodies against Arl13b and Centrin and DAPI . To quantify the percentage of ciliated cells ( J ) , > 80 cells were analyzed for each experiment . Scale bar , 2 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 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 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

(I, J) Immunofluorescence images (I) and quantification of the percentage of ciliated cells (J, n = 3 independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs, cultured in serum-free medium, and stained with antibodies against Arl13b and Centrin and DAPI. To quantify the percentage of ciliated cells (J), >80 cells were analyzed for each experiment. Scale bar, 2 µm.


( K , L ) Immunofluorescence images ( K ) and quantification of the percentage of ciliated cells ( L , n = 3 independent experiments ) for RPE1 cells transfected with control or ENKD1 siRNAs and plasmids expressing GFP , GFP - ENKD1 , GFP - ENKD1 - N , GFP - ENKD1 - M , or GFP - ENKD1 - C , cultured in serum - free medium , and stained with the antibody against acetylated α - tubulin and DAPI . The siRNA - resistant forms of ENKD1 were used for these rescue experiments . To quantify the percentage of ciliated cells ( L ) , > 120 cells were analyzed for each experiment . Scale bar , 3 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 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 B-CONTROLLED_VAR 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 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 O O 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, L) Immunofluorescence images (K) and quantification of the percentage of ciliated cells (L, n = 3 independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs and plasmids expressing GFP, GFP-ENKD1, GFP-ENKD1-N, GFP-ENKD1-M, or GFP-ENKD1-C, cultured in serum-free medium, and stained with the antibody against acetylated α-tubulin and DAPI. The siRNA-resistant forms of ENKD1 were used for these rescue experiments. To quantify the percentage of ciliated cells (L), >120 cells were analyzed for each experiment. Scale bar, 3 µm.


( A ) Immunofluorescence images of RPE1 cells cultured in normal serum medium , and stained with antibodies against ENKD1 and Centrin and DAPI . Scale bar , 1 µm .

O 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 O O O O O O O O

(A) Immunofluorescence images of RPE1 cells cultured in normal serum medium, and stained with antibodies against ENKD1 and Centrin and DAPI. Scale bar, 1 µm.


( B ) Immunofluorescence images and fluorescence intensity profiles of ENKD1 and CEP164 in serum - starved RPE1 cells . The arrow marks the centriole subjected to the line scan . Scale bar , 1 µm .

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 O O O O

(B) Immunofluorescence images and fluorescence intensity profiles of ENKD1 and CEP164 in serum-starved RPE1 cells. The arrow marks the centriole subjected to the line scan. Scale bar, 1 µm.


( C ) Immunofluorescence images and fluorescence intensity profiles of ENKD1 and Ninein in serum - starved RPE1 cells . The arrow marks the centriole subjected to the line scan . Scale bar , 2 µm .

(C) Immunofluorescence images and fluorescence intensity profiles of ENKD1 and Ninein in serum-starved RPE1 cells. The arrow marks the centriole subjected to the line scan. Scale bar, 2 µm.


( D - F ) 3D - SIM super - resolution images ( D ) , quantification of the outer toroid diameters ( E , n = 20 cells from three independent experiments ) , and a model illustrating the relative positions of γ - tubulin , Ninein , ENKD1 , CEP164 , CEP912 , and Centrin ( F ) within a centriole . RPE1 cells were serum - starved and stained with antibodies against the indicated proteins . Scale bar , 1 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 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 B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_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

(D-F) 3D-SIM super-resolution images (D), quantification of the outer toroid diameters (E, n = 20 cells from three independent experiments), and a model illustrating the relative positions of γ-tubulin, Ninein, ENKD1, CEP164, CEP912, and Centrin (F) within a centriole. RPE1 cells were serum-starved and stained with antibodies against the indicated proteins. Scale bar, 1 µm.


( G ) Immunofluorescence images of RPE1 cells cultured in normal serum or serum - free medium , and stained with antibodies against ENKD1 and γ - tubulin and DAPI . Scale bar , 1 µm .

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

(G) Immunofluorescence images of RPE1 cells cultured in normal serum or serum-free medium, and stained with antibodies against ENKD1 and γ-tubulin and DAPI. Scale bar, 1 µm.


( H ) Immunofluorescence images of RPE1 cells cultured in serum - free medium , and stained with antibodies against ENKD1 and acetylated α - tubulin and DAPI . Scale bar , 1 µm .

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

(H) Immunofluorescence images of RPE1 cells cultured in serum-free medium, and stained with antibodies against ENKD1 and acetylated α-tubulin and DAPI. Scale bar, 1 µm.


( I ) Immunofluorescence images of RPE1 cells transfected with GFP - ENKD1 or GFP vector , cultured in a serum - starved condition , and stained with the antibody against acetylated α - tubulin and DAPI . Scale bar , 1 µm .

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

(I) Immunofluorescence images of RPE1 cells transfected with GFP-ENKD1 or GFP vector, cultured in a serum-starved condition, and stained with the antibody against acetylated α-tubulin and DAPI. Scale bar, 1 µm.


( K ) 3D - SIM images of ENKD1 localization at the centrosome in RPE1 cells cultured in serum - free medium ( 0 , 12 , 24 , or 48 h of serum starvation ) , and stained with antibodies against ENKD1 and acetylated α - tubulin . Scale bar , 1 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 O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O

(K) 3D-SIM images of ENKD1 localization at the centrosome in RPE1 cells cultured in serum-free medium (0, 12, 24, or 48 h of serum starvation), and stained with antibodies against ENKD1 and acetylated α-tubulin. Scale bar, 1 µm.


Immunofluorescence images ( B ) for RPE1 cells transfected with control or ENKD1 siRNAs , cultured in serum - free medium , and stained with antibodies against CP110 and acetylated α - tubulin and DAPI .

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

Immunofluorescence images (B) for RPE1 cells transfected with control or ENKD1 siRNAs, cultured in serum-free medium, and stained with antibodies against CP110 and acetylated α-tubulin and DAPI.


quantification of the percentage of cells with one CP110 dot ( C , n = 3 independent experiments ) for RPE1 cells transfected with control or ENKD1 siRNAs To quantify the percentage of cells with one CP110 dot ( C ) , > 120 cells were analyzed for each experiment .

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 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

quantification of the percentage of cells with one CP110 dot (C, n = 3 independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs To quantify the percentage of cells with one CP110 dot (C), >120 cells were analyzed for each experiment.


( D , E ) Immunofluorescence images ( D ) and quantification of the percentage of cells with one CEP97 dot ( E , n = 3 independent experiments ) for RPE1 cells transfected with control or ENKD1 siRNAs , cultured in serum - free medium , and stained with antibodies against CEP97 and acetylated α - tubulin and DAPI . To quantify the percentage of cells with one CEP97 dot ( E ) , > 150 cells were analyzed for each experiment . Scale bar , 1 µm .

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

(D, E) Immunofluorescence images (D) and quantification of the percentage of cells with one CEP97 dot (E, n = 3 independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs, cultured in serum-free medium, and stained with antibodies against CEP97 and acetylated α-tubulin and DAPI. To quantify the percentage of cells with one CEP97 dot (E), >150 cells were analyzed for each experiment. Scale bar, 1 µm.


( F - H ) Immunoblot analysis ( F ) , immunofluorescence images ( G ) , and quantification of the percentage of cells with one CP110 dot ( H , n = 3 independent experiments ) for RPE1 cells transfected with control or ENKD1 siRNAs , cultured in serum - free medium for 120 h , and stained with antibodies against CP110 and acetylated α - tubulin and DAPI . To quantify the percentage of cells with one CP110 dot ( C ) , > 80 cells were analyzed for each experiment . Scale bar , 1 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-MEASURED_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 B-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-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 O O O O O O O O O O

(F-H) Immunoblot analysis (F), immunofluorescence images (G), and quantification of the percentage of cells with one CP110 dot (H, n = 3 independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs, cultured in serum-free medium for 120 h, and stained with antibodies against CP110 and acetylated α-tubulin and DAPI. To quantify the percentage of cells with one CP110 dot (C), >80 cells were analyzed for each experiment. Scale bar, 1 µm.


( A - C ) Immunoblot analysis ( A ) , immunofluorescence images ( B ) and quantification of the percentage of cells with one CP110 dot ( C , n = 3 independent experiments ) for RPE1 cells transfected with the indicated siRNAs , cultured in serum - free medium for 48 h , and stained with antibodies against CP110 and acetylated α - tubulin and DAPI . To quantify the percentage of cells with one CP110 dot ( C ) , > 180 cells were analyzed for each experiment . Scale bar , 1 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 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 B-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-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 O O O O O O O O O O

(A-C) Immunoblot analysis (A), immunofluorescence images (B) and quantification of the percentage of cells with one CP110 dot (C, n = 3 independent experiments) for RPE1 cells transfected with the indicated siRNAs, cultured in serum-free medium for 48 h, and stained with antibodies against CP110 and acetylated α-tubulin and DAPI. To quantify the percentage of cells with one CP110 dot (C), >180 cells were analyzed for each experiment. Scale bar, 1 µm.


( D ) Immunoblot analysis of CP110 and β - actin in RPE1 cells transfected with control or CP110 siRNAs and cultured in serum - free medium for 48 h .

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

(D) Immunoblot analysis of CP110 and β-actin in RPE1 cells transfected with control or CP110 siRNAs and cultured in serum-free medium for 48 h.


( E , F ) Immunofluorescence images ( E ) and quantification of the percentage of ciliated cells ( F , n = 3 independent experiments ) for RPE1 cells transfected with the indicated siRNAs , cultured in a serum - starved condition , and stained with antibodies against γ - tubulin and acetylated α - tubulin and DAPI . To quantify the percentage of ciliated cells ( F ) , > 120 cells were analyzed for each experiment . Scale bar , 10 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 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 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

(E, F) Immunofluorescence images (E) and quantification of the percentage of ciliated cells (F, n = 3 independent experiments) for RPE1 cells transfected with the indicated siRNAs, cultured in a serum-starved condition, and stained with antibodies against γ-tubulin and acetylated α-tubulin and DAPI. To quantify the percentage of ciliated cells (F), >120 cells were analyzed for each experiment. Scale bar, 10 µm.


( G , H ) Immunoprecipitation and immunoblotting ( G ) and quantification ( H , n = 3 mice ) showing the CP110 - CEP97 interaction in the retinal tissues of wild - type and Enkd1 knockout mice . The intensity of each CEP97 band was normalized to that of the β - actin band .

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

(G, H) Immunoprecipitation and immunoblotting (G) and quantification (H, n = 3 mice) showing the CP110-CEP97 interaction in the retinal tissues of wild-type and Enkd1 knockout mice. The intensity of each CEP97 band was normalized to that of the β-actin band.


( I , J ) Immunoprecipitation and immunoblotting ( I ) and quantification ( J , n = 3 independent experiments ) showing the CP110 - CEP97 interaction in serum - starved RPE1 cells transfected with control or ENKD1 siRNAs . The intensity of each CEP97 band was normalized to that of the β - actin band .

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-MEASURED_VAR 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

(I, J) Immunoprecipitation and immunoblotting (I) and quantification (J, n = 3 independent experiments) showing the CP110-CEP97 interaction in serum-starved RPE1 cells transfected with control or ENKD1 siRNAs. The intensity of each CEP97 band was normalized to that of the β-actin band.


( A ) Immunoprecipitation and immunoblotting showing the interaction between endogenous ENKD1 and CP110 in mouse retinal tissues . Immunoprecipitation was performed with the anti - ENKD1 ( upper panel ) or anti - CP110 ( lower panel ) antibody or the corresponding control IgG .

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

(A) Immunoprecipitation and immunoblotting showing the interaction between endogenous ENKD1 and CP110 in mouse retinal tissues. Immunoprecipitation was performed with the anti-ENKD1 (upper panel) or anti-CP110 (lower panel) antibody or the corresponding control IgG.


( B - D ) Immunoprecipitation and immunoblotting showing the interaction of GFP - ENKD1 with Flag - CP110 ( B and upper panel of D ) or HA - CP110 ( C and bottom panel of D ) in HEK293T cells . Immunoprecipitation was performed with antibodies against GFP ( B and C ) , Flag ( upper panel of D ) , or HA ( bottom panel of D ) .

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 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

(B-D) Immunoprecipitation and immunoblotting showing the interaction of GFP-ENKD1 with Flag-CP110 (B and upper panel of D) or HA-CP110 (C and bottom panel of D) in HEK293T cells. Immunoprecipitation was performed with antibodies against GFP (B and C), Flag (upper panel of D), or HA (bottom panel of D).


( E ) Identification of the domains of ENKD1 mediating its interaction with CP110 .

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

(E) Identification of the domains of ENKD1 mediating its interaction with CP110.


( F ) Identification of the domains of CP110 mediating its interaction with ENKD1 .

(F) Identification of the domains of CP110 mediating its interaction with ENKD1.


( H ) GST pull - down showing the interaction of purified GST - CP110 - N with purified His - ENKD1 - M .

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

(H) GST pull-down showing the interaction of purified GST-CP110-N with purified His-ENKD1-M.


( I , J ) Immunoprecipitation and immunoblotting ( I ) and quantification ( J , n = 3 independent experiments ) showing the interaction of HA - CP110 with Flag - CEP97 in HEK293T cells transfected with the indicated plasmids . The intensity of each Flag - CEP97 band was normalized to that of the β - actin band .

O O O O 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 B-MEASURED_VAR 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

(I, J) Immunoprecipitation and immunoblotting (I) and quantification (J, n = 3 independent experiments) showing the interaction of HA-CP110 with Flag-CEP97 in HEK293T cells transfected with the indicated plasmids. The intensity of each Flag-CEP97 band was normalized to that of the β-actin band.


A - D Mesenteric blood ( PECAM1 , red ) and lymphatic vessels ( LYVE1 , green ; PROX1 , gray ) . Asterisks indicate lymphatic valves , arrow indicates a lymphatic vessel stub , and arrowhead indicates an isolated lymphatic vessel fragmentE - H Blood vessels ( PECAM1 , green ) and lymphatic vessels ( VEGFR - 3 , red ) in the small intestinal wall .

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

A-D Mesenteric blood (PECAM1, red) and lymphatic vessels (LYVE1, green; PROX1, gray). Asterisks indicate lymphatic valves, arrow indicates a lymphatic vessel stub, and arrowhead indicates an isolated lymphatic vessel fragmentE-H Blood vessels (PECAM1, green) and lymphatic vessels (VEGFR-3, red) in the small intestinal wall.


I , J Detection of chylous ascites ( arrows ) in the VCiΔR26 mice at P6 .

I, J Detection of chylous ascites (arrows) in the VCiΔR26 mice at P6.


K , L LYVE1 staining of intestinal lymphatic vessels at P6 .

K, L LYVE1 staining of intestinal lymphatic vessels at P6.


M - T LYVE1 staining of lymphatic vessels in the intestinal wall in adult mice . Genotypes and deletion lengths are indicated in ( U ) . U Quantification of LYVE1 areas in ( M - T ) . Length of the i ∆ Vegfc gene deletion is indicated in months ( mo ) , and Vegfd indicates the VEGF - D genotype . Data are represented as mean ± SEM . Significant differences were determined using one - way ANOVA and Bonferroni post hoc analysis compared to WT intestine represented in ( M ) . * P = 0 . 003 , * * P = 0 . 001 , * * * P = 0 . 0008 , ♯ P = 0 . 0002 , § P = 0 . 0001 .

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 B-MEASURED_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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

M-T LYVE1 staining of lymphatic vessels in the intestinal wall in adult mice. Genotypes and deletion lengths are indicated in (U).U Quantification of LYVE1 areas in (M-T). Length of the i∆Vegfc gene deletion is indicated in months (mo), and Vegfd indicates the VEGF-D genotype. Data are represented as mean ± SEM. Significant differences were determined using one-way ANOVA and Bonferroni post hoc analysis compared to WT intestine represented in (M). *P = 0.003, **P = 0.001, ***P = 0.0008, ♯P = 0.0002, §P = 0.0001.


Overview of the small intestine cross section stained with nuclear red and highlighting the location of higher magnification images in ( B - F ) ; ( I ) for the entire villus and ( II ) for the villus base . β - Gal staining pattern of the villus in wild - type ( Ctrl ) , Vegfc / LacZ ( VC ) , Vegfr3 / LacZ ( VR - 3 ) , and Vegfr2 / LacZ ( VR - 2 ) mice . Higher magnification images representing β - Gal staining of the villus base in Vegfc / LacZmice . Data information : Arrows indicate the VEGF - C expression in arterialSMC , arrowheads indicate the VEGF - C expression in SMCfibers in the villus , and asterisks highlight the VEGF - C expression in circular smooth muscle cell layer of the intestinal wall . Scale bars : 50 μm , except ( C ) inset 25 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 O O O O O O O O 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 B-CONTROLLED_VAR 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 B-CONTROLLED_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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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

Overview of the small intestine cross section stained with nuclear red and highlighting the location of higher magnification images in (B-F); (I) for the entire villus and (II) for the villus base. β-Gal staining pattern of the villus in wild-type (Ctrl), Vegfc/LacZ (VC), Vegfr3/LacZ (VR-3), and Vegfr2/LacZ (VR-2) mice.Higher magnification images representing β-Gal staining of the villus base in Vegfc/LacZmice.Data information: Arrows indicate the VEGF-C expression in arterialSMC, arrowheads indicate the VEGF-C expression in SMCfibers in the villus, and asterisks highlight the VEGF-C expression in circular smooth muscle cell layer of the intestinal wall. Scale bars: 50 μm, except (C) inset 25 μm.


Vegfc / LacZβ - Gal staining reaction with smooth muscle actin ( SMA ) peroxidase staining . Data information : Arrows indicate the VEGF - C expression in arterialSMC , arrowheads indicate the VEGF - C expression in SMCfibers in the villus , and asterisks highlight the VEGF - C expression in circular smooth muscle cell layer of the intestinal wall . Scale bars : 50 μm , except ( C ) inset 25 μm .

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

Vegfc/LacZβ-Gal staining reaction with smooth muscle actin (SMA) peroxidase staining.Data information: Arrows indicate the VEGF-C expression in arterialSMC, arrowheads indicate the VEGF-C expression in SMCfibers in the villus, and asterisks highlight the VEGF-C expression in circular smooth muscle cell layer of the intestinal wall. Scale bars: 50 μm, except (C) inset 25 μm.


Vegfc / LacZβ - Gal staining and LYVE1 peroxidase staining . Data information : Arrows indicate the VEGF - C expression in arterialSMC , arrowheads indicate the VEGF - C expression in SMCfibers in the villus , and asterisks highlight the VEGF - C expression in circular smooth muscle cell layer of the intestinal wall . Scale bars : 50 μm , except ( C ) inset 25 μm .

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

Vegfc/LacZβ-Gal staining and LYVE1 peroxidase staining.Data information: Arrows indicate the VEGF-C expression in arterialSMC, arrowheads indicate the VEGF-C expression in SMCfibers in the villus, and asterisks highlight the VEGF-C expression in circular smooth muscle cell layer of the intestinal wall. Scale bars: 50 μm, except (C) inset 25 μm.


Surface image of Vegfc / LacZβ - Gal - stained intestine ( left ) and cross section counterstaining with PECAM1 ( right ) . Data information : Arrows indicate the VEGF - C expression in arterialSMC , arrowheads indicate the VEGF - C expression in SMCfibers in the villus , and asterisks highlight the VEGF - C expression in circular smooth muscle cell layer of the intestinal wall . Scale bars : 50 μm , except ( C ) inset 25 μm .

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

Surface image of Vegfc/LacZβ-Gal-stained intestine (left) and cross section counterstaining with PECAM1 (right).Data information: Arrows indicate the VEGF-C expression in arterialSMC, arrowheads indicate the VEGF-C expression in SMCfibers in the villus, and asterisks highlight the VEGF-C expression in circular smooth muscle cell layer of the intestinal wall. Scale bars: 50 μm, except (C) inset 25 μm.


Immunofluorescence staining of lacteal lymphatic vessel ( LYVE1 ) , blood capillaries ( PECAM1 ) , and smooth muscle cells ( SMA ) . Data information : Arrows indicate the VEGF - C expression in arterialSMC , arrowheads indicate the VEGF - C expression in SMCfibers in the villus , and asterisks highlight the VEGF - C expression in circular smooth muscle cell layer of the intestinal wall . Scale bars : 50 μm , except ( C ) inset 25 μm .

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 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

Immunofluorescence staining of lacteal lymphatic vessel (LYVE1), blood capillaries (PECAM1), and smooth muscle cells (SMA).Data information: Arrows indicate the VEGF-C expression in arterialSMC, arrowheads indicate the VEGF-C expression in SMCfibers in the villus, and asterisks highlight the VEGF-C expression in circular smooth muscle cell layer of the intestinal wall. Scale bars: 50 μm, except (C) inset 25 μm.


Two - month - old mice received tamoxifen and were fed on high - fat diet ( HFD ) for seven weeks before analysis . Body weight change during seven weeks of HFD , expressed as average fold change in comparison with the starting weight . n = 16 , WT ; n = 6 , Vegfd − / − ; n = 16 , VCiΔR26 ; n = 5 , Vegfd − / − ; VCiΔR26 . Body weight comparisons at seven weeks of HFD . Significant differences were determined using one - way ANOVA and Bonferroni post hoc analysis compared to WT . * P = 0 . 004 ; * * P = 0 . 003 . n = 16 , WT ; n = 6 , Vegfd − / − ; n = 16 , VCiΔR26 ; n = 5 , Vegfd − / − ; VCiΔR26 .

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

Two-month-old mice received tamoxifen and were fed on high-fat diet (HFD) for seven weeks before analysis.Body weight change during seven weeks of HFD, expressed as average fold change in comparison with the starting weight. n = 16, WT; n = 6, Vegfd−/−; n = 16, VCiΔR26; n = 5, Vegfd−/−; VCiΔR26.Body weight comparisons at seven weeks of HFD. Significant differences were determined using one-way ANOVA and Bonferroni post hoc analysis compared to WT. *P = 0.004; **P = 0.003. n = 16, WT; n = 6, Vegfd−/−; n = 16, VCiΔR26; n = 5, Vegfd−/−; VCiΔR26.


Two - month - old mice received tamoxifen and were fed on high - fat diet ( HFD ) for seven weeks before analysis . Glucose tolerance test ( GTT ) after six weeks of HFD . Significant differences were determined using unpaired two - tailed t - test . * P = 0 . 014 ; * * P = 0 . 041 . n = 5 , WT ; n = 6 , VCiΔR26 .

O 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 O O O O O O O O

Two-month-old mice received tamoxifen and were fed on high-fat diet (HFD) for seven weeks before analysis. Glucose tolerance test (GTT) after six weeks of HFD. Significant differences were determined using unpaired two-tailed t-test. *P = 0.014; **P = 0.041. n = 5, WT; n = 6, VCiΔR26.


Two - month - old mice received tamoxifen and were fed on high - fat diet ( HFD ) for seven weeks before analysis . Total fatweight , fat percentage from body composition measurements after six weeks of HFD , and weights of visceral fat ( VF ) and subcutaneous fat ( SF ) at the time of necropsy . Significant differences were determined using unpaired two - tailed t - test . * P = 0 . 006 ; * * P = 0 . 001 ; # P = 0 . 008 ; § P = 0 . 006 . n = 4 in 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Two-month-old mice received tamoxifen and were fed on high-fat diet (HFD) for seven weeks before analysis. Total fatweight, fat percentage from body composition measurements after six weeks of HFD, and weights of visceral fat (VF) and subcutaneous fat (SF) at the time of necropsy. Significant differences were determined using unpaired two-tailed t-test. *P = 0.006; **P = 0.001; #P = 0.008; §P = 0.006. n = 4 in each group.


Two - month - old mice received tamoxifen and were fed on high - fat diet ( HFD ) for seven weeks before analysis . Food consumption during the fifth week of HFD . n = 9 , WT ; n = 10 , VCiΔR26 .

O 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

Two-month-old mice received tamoxifen and were fed on high-fat diet (HFD) for seven weeks before analysis. Food consumption during the fifth week of HFD. n = 9, WT; n = 10, VCiΔR26.


Two - month - old mice received tamoxifen and were fed on high - fat diet ( HFD ) for seven weeks before analysis . Whole - mount immunofluorescence staining of blood ( PECAM1 , red ) and lymphatic vessels ( LYVE1 , green ) in intestinal villi and intestinal wall . Quantification of the lacteal and villus length ( solid and striped color bars , respectively ) and the intestinal wallLYVE1 + area percentage from images represented in ( F ) . Significant differences were determined using unpaired two - tailed t - test . * P = 0 . 0002 ; * * P = 0 . 00007 . n = 5 , WT ; n = 6 , VCiΔR26 .

O O O O O O O O O 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 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

Two-month-old mice received tamoxifen and were fed on high-fat diet (HFD) for seven weeks before analysis.Whole-mount immunofluorescence staining of blood (PECAM1, red) and lymphatic vessels (LYVE1, green) in intestinal villi and intestinal wall.Quantification of the lacteal and villus length (solid and striped color bars, respectively) and the intestinal wallLYVE1+ area percentage from images represented in (F). Significant differences were determined using unpaired two-tailed t-test. *P = 0.0002; **P = 0.00007. n = 5, WT; n = 6, VCiΔR26.


Two - month - old mice received tamoxifen and were fed on high - fat diet ( HFD ) for seven weeks before analysis . Free fatty acid ( FFA ) and cholesterol measurements from the feces after six weeks of HFD . Significant differences were determined using unpaired two - tailed t - test . * P = 0 . 001 ; * * P = 0 . 007 . n = 5 , WT ; n = 6 , VCiΔR26 .

O O 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 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 O

Two-month-old mice received tamoxifen and were fed on high-fat diet (HFD) for seven weeks before analysis. Free fatty acid (FFA) and cholesterol measurements from the feces after six weeks of HFD. Significant differences were determined using unpaired two-tailed t-test. *P = 0.001; **P = 0.007. n = 5, WT; n = 6, VCiΔR26.


A . Relative antiviral activity of IFNα ( circles ) or IFNλ ( triangles ) . AEC cultures were stimulated for 4hrs with stated IFN at specified concentrations ( ng / ml ) and induction of indicated ISGs was assessed by qPCR ( data shown is representative of four independent experiments , n = 3 - 4 ) .

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

A. Relative antiviral activity of IFNα (circles) or IFNλ (triangles). AEC cultures were stimulated for 4hrs with stated IFN at specified concentrations (ng/ml) and induction of indicated ISGs was assessed by qPCR (data shown is representative of four independent experiments, n=3-4).


B , C . Mice were pretreated with equivalent doses of IFNα ( 1 . 45μg / 50μl ) or IFNλ ( 2 . 6μg / 50μl ) or Veh Ctrl ( squares , 50μl PBS ) 24hrs prior to infection with PR8 ; weight loss and survival was assessed throughout infection ( B ) , and viral load ( C ) assessed at 4dpi ( data shown is representative of two independent experiments , n = 8 - 10 ( B ) , n = 3 ( C ) ) .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Mice were pretreated with equivalent doses of IFNα (1.45μg/50μl) or IFNλ (2.6μg/50μl) or Veh Ctrl (squares, 50μl PBS) 24hrs prior to infection with PR8; weight loss and survival was assessed throughout infection (B), and viral load (C) assessed at 4dpi (data shown is representative of two independent experiments, n=8-10 (B), n=3 (C)).


D , E . Mice were infected with PR8 and treated with equivalent doses of IFNα or IFNλ or Veh Ctrl at days 2 , 4 and 5 post infection ; survival and weight loss was monitored ( D , data pooled from 4 independent experiments , n = 12 - 29 ) ( data representative of two independent experiments , n = 3 - 5 ) .

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 O O O O O O O O O O O O O O O O O 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, E. Mice were infected with PR8 and treated with equivalent doses of IFNα or IFNλ or Veh Ctrl at days 2, 4 and 5 post infection; survival and weight loss was monitored (D, data pooled from 4 independent experiments, n=12-29) (data representative of two independent experiments, n=3-5).


D , E . Mice were infected with PR8 and treated with equivalent doses of IFNα or IFNλ or Veh Ctrl at days 2 , 4 and 5 post infection ; viral load assessed at 4dpi ( E ) ( data representative of two independent experiments , n = 3 - 5 ) .

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 O O O 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, E. Mice were infected with PR8 and treated with equivalent doses of IFNα or IFNλ or Veh Ctrl at days 2, 4 and 5 post infection; viral load assessed at 4dpi (E) (data representative of two independent experiments, n=3-5).


A , B . Mice were infected with PR8 and treated with IFNα ( circles , 1 . 45μg / 50μl ) , IFNλ ( triangles , 2 . 6μg / 50μl ) or Veh Ctrl ( squares ) as previously stated . Concentrations of stated proinflammatory cytokines in BAL fluid was measured by multiplex cytokine assay ( A ) ( data shown is representative of two independent experiments , n = 2 - 6 ) .

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 O O O O O O O O O O O O O O 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, B. Mice were infected with PR8 and treated with IFNα (circles, 1.45μg/50μl), IFNλ (triangles, 2.6μg/50μl) or Veh Ctrl (squares) as previously stated. Concentrations of stated proinflammatory cytokines in BAL fluid was measured by multiplex cytokine assay (A) (data shown is representative of two independent experiments, n=2-6).


A , B . Mice were infected with PR8 and treated with IFNα ( circles , 1 . 45μg / 50μl ) , IFNλ ( triangles , 2 . 6μg / 50μl ) or Veh Ctrl ( squares ) as previously stated . Concentrations of stated proinflammatory cytokines in BAL fluid was measured by flow cytometry quantification of pDCs and Inflammatory Monocytes in the lung was performed ( B ) ( data shown is representative of two independent experiments , n = 2 - 6 ) .

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 O O O O O O O O O O O O O O O O O O O O O O O O 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, B. Mice were infected with PR8 and treated with IFNα (circles, 1.45μg/50μl), IFNλ (triangles, 2.6μg/50μl) or Veh Ctrl (squares) as previously stated. Concentrations of stated proinflammatory cytokines in BAL fluid was measured by flow cytometry quantification of pDCs and Inflammatory Monocytes in the lung was performed (B) (data shown is representative of two independent experiments, n=2-6).


C , D . Lung sections from control and infected mice treated as indicated , were stained by TUNEL for apoptotic cells at 6dpi . Quantification of TUNEL + cells in whole lung slides by Icy Spot Detector ( ICY - R3M2Y2 ) ( C ) ( data shown is pooled from three independent experiments , n = 3 - 8 ) . Red arrowheads indicate TUNEL signal ( D ) . Scale bar , 200 μM ( data shown is representative of two independent experiments , n = 3 - 4 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Lung sections from control and infected mice treated as indicated, were stained by TUNEL for apoptotic cells at 6dpi. Quantification of TUNEL+ cells in whole lung slides by Icy Spot Detector (ICY-R3M2Y2) (C) (data shown is pooled from three independent experiments, n=3-8). Red arrowheads indicate TUNEL signal (D). Scale bar, 200 μM (data shown is representative of two independent experiments, n=3-4).


A , B . IL - 6 , IP - 10 and MCP - 1 concentrations were measured by multiplex cytokine assay in AEC culture supernatants ( A ) at 24hrs post stimulation with IFNα4 ( 0 . 725ng / ml ) or IFNλ2 ( 1 . 3ng / ml ) or LPS ( AEC only ) ( data shown is representative of two independent experiments , n = 3 - 6 ) .

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

A, B. IL-6, IP-10 and MCP-1 concentrations were measured by multiplex cytokine assay in AEC culture supernatants (A) at 24hrs post stimulation with IFNα4 (0.725ng/ml) or IFNλ2 (1.3ng/ml) or LPS (AEC only) (data shown is representative of two independent experiments, n=3-6).


A , B . IL - 6 , IP - 10 and MCP - 1 concentrations were measured by multiplex cytokine assay in Macrophage , pDC and cDC culture supernatants ( B ) at 24hrs post stimulation with IFNα4 ( 0 . 725ng / ml ) or IFNλ2 ( 1 . 3ng / ml ) ( data shown is representative of two independent experiments , n = 3 - 6 ) .

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

A, B. IL-6, IP-10 and MCP-1 concentrations were measured by multiplex cytokine assay in Macrophage, pDC and cDC culture supernatants (B) at 24hrs post stimulation with IFNα4 (0.725ng/ml) or IFNλ2 (1.3ng/ml) (data shown is representative of two independent experiments, n=3-6).


C . BAL samples taken from mice treated with IFNα , IFNλ or Veh Ctrl at specified time points ( data shown is representative of two independent experiments , n = 5 - 6 ) .

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

C. BAL samples taken from mice treated with IFNα, IFNλ or Veh Ctrl at specified time points (data shown is representative of two independent experiments, n=5-6).


Mice were treated with IFNα ( 1 . 45μg / 50μl ) , IFNλ ( 2 . 6μg / 50μl ) or Veh Ctrl ( 50μl PBS ) , and whole lungs were taken at 18hrs post treatment for global analysis by Illumina . SingleColor . Mouse WG - 6 _ V2 _ 0 _ R0 _ 1127 microarrays . Samples ( n = 5 ) were normalized to the median of the vehicle control group and filtered for a fold change of 1 . 5 , yielding 553 genes differently regulated between treatments ( One way ANOVA , P < 0 . 01 , Benjamini - Hochberg multiple test correction ) , of which 429 genes are upregulated . K means clustering revealed six gene clusters , one of which encompassed genes primarily induced by IFNα4 ( A ) , while the remaining clusters contained genes upregulated by both IFNα4 and IFNλ2 ( B ) . The two clusters of genes were analysed by Ingenuity Pathway Analysis ( IPA ) ( C , D ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-CONTROLLED_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

Mice were treated with IFNα (1.45μg/50μl), IFNλ (2.6μg/50μl) or Veh Ctrl (50μl PBS), and whole lungs were taken at 18hrs post treatment for global analysis by Illumina.SingleColor.Mouse WG-6_V2_0_R0_1127 microarrays. Samples (n=5) were normalized to the median of the vehicle control group and filtered for a fold change of 1.5, yielding 553 genes differently regulated between treatments (One way ANOVA, P<0.01, Benjamini-Hochberg multiple test correction), of which 429 genes are upregulated. K means clustering revealed six gene clusters, one of which encompassed genes primarily induced by IFNα4 (A), while the remaining clusters contained genes upregulated by both IFNα4 and IFNλ2 (B). The two clusters of genes were analysed by Ingenuity Pathway Analysis (IPA) (C,D).


A . HumanAEC cultures were stimulated for 4hrs with IFNα ( circles ) or IFNλ ( triangles ) at specified concentrations then assessed for stated ISG induction by qPCR ( data is representative of 2 independent experiments , n = 3 ) .

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 O O O O O O O O O O

A. HumanAEC cultures were stimulated for 4hrs with IFNα (circles) or IFNλ (triangles) at specified concentrations then assessed for stated ISG induction by qPCR (data is representative of 2 independent experiments, n=3).


B , C . ISG induction in humanPBMCs was assessed at 4 and 24hrs post IFNα ( 21 U / ml ) or IFNλ ( 1 . 2 ng / ml ) stimulation ( B ) ( data shown is pooled from 6 independent donors ) .

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 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, C. ISG induction in humanPBMCs was assessed at 4 and 24hrs post IFNα (21 U/ml) or IFNλ (1.2 ng/ml) stimulation (B) (data shown is pooled from 6 independent donors).


B , C . PBMC proinflammatory cytokine secretion was measured by multiplex cytokine assay at 4 and 24hrs post stimulation with IFNα or IFNλ ( C ) ( data shown is pooled from 6 independent donors ) .

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 O O O O O

B, C. PBMC proinflammatory cytokine secretion was measured by multiplex cytokine assay at 4 and 24hrs post stimulation with IFNα or IFNλ (C) (data shown is pooled from 6 independent donors).


B Box plots of the Em / Ad expression ratios of GATA6 ( left ) and NKX2 - 1 ( right ) in FFPE lung tissue sections from controls ( Ctrl , n = 61 ) or lung cancer ( LC , n = 51 ) patients . Isoform - specific expression of the indicated genes was analyzed by qRT - PCR after total RNA isolation from tissue samples . Each point represents one sample .

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

B Box plots of the Em/Ad expression ratios of GATA6 (left) and NKX2-1 (right) in FFPE lung tissue sections from controls (Ctrl, n=61) or lung cancer (LC, n=51) patients. Isoform-specific expression of the indicated genes was analyzed by qRT-PCR after total RNA isolation from tissue samples. Each point represents one sample.


C Box plots of Em / Ad of GATA6 ( top ) or NKX2 - 1 ( bottom ) show that high Em / Ad ratios in LC samples are maintained among ethnic groups ( left ) and gender ( right ) . GER , samples collected in Germany ; MEX , samples collected in Mexico .

O O O O O O O O B-MEASURED_VAR 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

C Box plots of Em/Ad of GATA6 (top) or NKX2-1 (bottom) show that high Em/Ad ratios in LC samples are maintained among ethnic groups (left) and gender (right). GER, samples collected in Germany; MEX, samples collected in Mexico.


D Box plots of Em / Ad of GATA6 ( left ) or NKX2 - 1 ( right ) in FFPE lung tissue sections from controls or LC patients . Samples were staged according to the TNM Classification ( UICC , 7th edition ) .

O O O O O O O O B-MEASURED_VAR 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

D Box plots of Em/Ad of GATA6 (left) or NKX2-1 (right) in FFPE lung tissue sections from controls or LC patients. Samples were staged according to the TNM Classification (UICC, 7th edition).


A Box plots of the Em / Ad expression ratios of GATA6 ( left ) and NKX2 - 1 ( right ) in EBCs from controls ( Ctrl , n = 65 ) or lung cancer ( LC , n = 48 ) patients ( training set ) . Each point represents one sample .

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

A Box plots of the Em/Ad expression ratios of GATA6 (left) and NKX2-1 (right) in EBCs from controls (Ctrl, n=65) or lung cancer (LC, n=48) patients (training set). Each point represents one sample.


( B ) Volcano plots showing differentially expressed genes between untreated and DLL4 & PDGFBB - treated mMuSCs , human myoblasts and hiMPs . Red dots represent genes which display a positive fold - change in expression upon treatment with DLL4 & PDGF - BB whilst violet dots represent genes which are significantly downregulated . Differentially expressed genes required a P value of ≤ 0 . 05 to be considered significant .

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

(B) Volcano plots showing differentially expressed genes between untreated and DLL4 & PDGFBB-treated mMuSCs, human myoblasts and hiMPs. Red dots represent genes which display a positive fold-change in expression upon treatment with DLL4 & PDGF-BB whilst violet dots represent genes which are significantly downregulated. Differentially expressed genes required a P value of ≤ 0.05 to be considered significant.


( C ) Heatmaps showing changes in expression of key myogenic ( MYOD , MYOGENIN ) , perivascular ( PDGFRB , NG2 , CD146 , ALPL ) and NOTCH target ( HEY1 , HES1 ) genes upon treatment with DLL4 & PDGF - BB in mMuSC - derived myoblasts ( left ) , human myoblasts ( middle ) and hiMPs ( right ) . Clustering was performed by genes / probes with Pearson correlation . Colour scale based on z - scores : red regions indicate high expression whilst blue regions indicate low expression . Dendrograms indicate the similarity of clusters as well as the orders in which clusters were assembled .

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

(C) Heatmaps showing changes in expression of key myogenic (MYOD, MYOGENIN), perivascular (PDGFRB, NG2, CD146, ALPL) and NOTCH target (HEY1, HES1) genes upon treatment with DLL4 & PDGF-BB in mMuSC-derived myoblasts (left), human myoblasts (middle) and hiMPs (right). Clustering was performed by genes/probes with Pearson correlation. Colour scale based on z-scores: red regions indicate high expression whilst blue regions indicate low expression. Dendrograms indicate the similarity of clusters as well as the orders in which clusters were assembled.