( C ) Micrographs showing dendrites from neurons transfected with Homer1c - BFP ( magenta ) , GFP - SP - shRNA ( gray ) and a rescue RFP - SP construct containing wild - type ( WT ) or mutated ( MUT ) 3 ' UTR ( geen ) and treated with TTX , or left untreated ( UT ) . Scale bar : 10 µm . ( D ) Percentage of SP + synapses for each condition ( 3 ' UTR - WT : UT , n = 26 , TTX , n = 28 ; 3 ' UTR - MUT : UT , n = 26 , TTX , n = 24 ; n indicates the number of cells , from 3 cultures ) . * * P < 0 . 01 , * * * P < 0 . 001 , ns , not significant , P > 0 . 05 ( two - way ANOVA test followed by Tukey ' s multiple comparison test ) . Data information : Data are represented as mean ± SEM .

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

(C) Micrographs showing dendrites from neurons transfected with Homer1c-BFP (magenta), GFP-SP-shRNA (gray) and a rescue RFP-SP construct containing wild-type (WT) or mutated (MUT) 3'UTR (geen) and treated with TTX, or left untreated (UT). Scale bar: 10 µm. (D) Percentage of SP+ synapses for each condition (3'UTR-WT: UT, n = 26, TTX, n = 28; 3'UTR-MUT: UT, n = 26, TTX, n = 24; n indicates the number of cells, from 3 cultures). **P < 0.01, ***P < 0.001, ns, not significant, P > 0.05 (two-way ANOVA test followed by Tukey's multiple comparison test). Data information: Data are represented as mean ± SEM.


( E ) Micrographs showing Homer1c - GFP ( magenta ) and immunostaining for surface AMPARs ( cyan ) and endogenous SP ( green ) in neurons transfected with or without 50 nM SP TSB - LNA and treated with TTX , or left untreated ( UT ) . Scale bar : 10 µm . ( F ) Percentage of SP + synapses for each condition ( Control : UT , n = 36 , TTX , n = 52 ; SP TSB - LNA : UT , n = 62 , TTX , n = 29 ; n indicates the number of cells from 3 cultures ) . * P < 0 . 05 , * * P < 0 . 01 , ns , not significant , P > 0 . 05 ( Kruskal - Wallis test followed by Dunn ' s multiple comparison test ) . ( G ) AMPAR synaptic fluorescence intensity for each condition , normalized to control untreated neurons ( Control : UT , n = 30 , TTX , n = 36 ; SP TSB - LNA : UT , n = 37 , TTX , n = 20 ; n indicates the number of cells from 2 cultures ) . * P < 0 . 05 , ns , not significant , P > 0 . 05 ( Kruskal - Wallis test followed by Dunn ' s multiple comparison test ) . Data information : Data are represented as mean ± SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O 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 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 B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Micrographs showing Homer1c-GFP (magenta) and immunostaining for surface AMPARs (cyan) and endogenous SP (green) in neurons transfected with or without 50 nM SP TSB-LNA and treated with TTX, or left untreated (UT). Scale bar: 10 µm. (F) Percentage of SP+ synapses for each condition (Control: UT, n = 36, TTX, n = 52; SP TSB-LNA: UT, n = 62, TTX, n = 29; n indicates the number of cells from 3 cultures). *P < 0.05, **P < 0.01, ns, not significant, P > 0.05 (Kruskal-Wallis test followed by Dunn's multiple comparison test). (G) AMPAR synaptic fluorescence intensity for each condition, normalized to control untreated neurons (Control: UT, n = 30, TTX, n = 36; SP TSB-LNA: UT, n = 37, TTX, n = 20; n indicates the number of cells from 2 cultures). *P < 0.05, ns, not significant, P > 0.05 (Kruskal-Wallis test followed by Dunn's multiple comparison test). Data information: Data are represented as mean ± SEM.


( A ) Micrographs showing puro - PLA staining of newly synthesized SP ( green ) in neurons immunostained for MAP - 2 ( magenta ) and DAPI ( blue ) and treated with TTX for 24 h or left untreated . The images on bottom panels show no staining in the absence of puromycin treatment or when omitting SP primary antibody . Scale bars : 30 µm ( large view ) , 5 µm ( insets ) .

O O O O O O O O O O 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

(A) Micrographs showing puro-PLA staining of newly synthesized SP (green) in neurons immunostained for MAP-2 (magenta) and DAPI (blue) and treated with TTX for 24 h or left untreated. The images on bottom panels show no staining in the absence of puromycin treatment or when omitting SP primary antibody. Scale bars: 30 µm (large view), 5 µm (insets).


( C ) Density of SP puro - PLA puncta , normalized to the untreated condition ( UT ) ( UT : n = 62 ; TTX : n = 57 , n represents the number of cells , from 3 cultures ) . * P < 0 . 05 ( Mann - Whitney test ) . ( D ) SP puro - PLA cluster fluorescence intensity normalized to the untreated condition ( UT ) ( UT : n = 62 ; TTX : n = 57 , n indicates the number of cells , from 3 cultures ) . * * * * P < 0 . 0001 ( Mann - Whitney test ) . Data information : Data are represented as mean ± SEM .

O O 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 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

(C) Density of SP puro-PLA puncta, normalized to the untreated condition (UT) (UT: n = 62; TTX: n = 57, n represents the number of cells, from 3 cultures). *P < 0.05 (Mann-Whitney test). (D) SP puro-PLA cluster fluorescence intensity normalized to the untreated condition (UT) (UT: n = 62; TTX: n = 57, n indicates the number of cells, from 3 cultures). ****P < 0.0001 (Mann-Whitney test). Data information: Data are represented as mean ± SEM.


( E ) Micrographs showing puro - PLA staining of newly synthesized SP ( green ) in neurons transfected with Homer1c - GFP ( magenta ) and treated with TTX for 24 h or left untreated . Scale bar : 5 µm . Arrowheads indicate puro - PLA + spines . ( F ) Percentage of SP puro - PLA + synapses ( UT , n = 26 , TTX , n = 33 ; n indicates the number of cells , from 2 cultures ) . * * P < 0 . 01 , ns , not significant ( Mann - Whitney test ) . ( G ) Homer1c - GFP integrated fluorescence intensity at SP puro - PLA - vs SP puro - PLA + synapses ( n = 32 cells , from 2 cultures ) . * * * * P < 0 . 0001 , ns , not significant ( Wilcoxon matched - pairs signed rank test ) . Data information : Data are represented as mean ± SEM .

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

(E) Micrographs showing puro-PLA staining of newly synthesized SP (green) in neurons transfected with Homer1c-GFP (magenta) and treated with TTX for 24 h or left untreated. Scale bar: 5 µm. Arrowheads indicate puro-PLA+ spines. (F) Percentage of SP puro-PLA+ synapses (UT, n = 26, TTX, n = 33; n indicates the number of cells, from 2 cultures). **P < 0.01, ns, not significant (Mann-Whitney test). (G) Homer1c-GFP integrated fluorescence intensity at SP puro-PLA- vs SP puro-PLA+ synapses (n = 32 cells, from 2 cultures). ****P < 0.0001, ns, not significant (Wilcoxon matched-pairs signed rank test). Data information: Data are represented as mean ± SEM.


( A ) Confocal images showing dendrites from CA3 pyramidal cells transfected with RFP - SP ( green ) containing wild - type ( WT ) or mutated ( MUT ) 3 ' UTR + SP - shRNA - BFP ( magenta ) . Arrowheads indicate SP + spines . Scale bar : 10 µm . ( B ) Graph plot showing spine head volume vs SP integrated intensity ( n = 5 cells ) . Each plot represents a single spine for which SP integrated intensity has been normalized to average SP intensity of SP + spines in the same neuron . Plots for SP - and SP + spines appear in grey and magenta , respectively . The black dotted line represents linear regression ( R2 = 0 . 25 , P = 0 . 0004 ) . ( C ) Paired data showing spine head volume for SP - vs SP + spines . Each pair of plot represents average values for SP - vs SP + spines for a given neuron . * P = 0 . 0031 ( two - tailed Student t test ) . Data information : Data are represented as mean ± SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Confocal images showing dendrites from CA3 pyramidal cells transfected with RFP-SP (green) containing wild-type (WT) or mutated (MUT) 3'UTR + SP-shRNA-BFP (magenta). Arrowheads indicate SP+ spines. Scale bar: 10 µm. (B) Graph plot showing spine head volume vs SP integrated intensity (n = 5 cells). Each plot represents a single spine for which SP integrated intensity has been normalized to average SP intensity of SP+ spines in the same neuron. Plots for SP- and SP+ spines appear in grey and magenta, respectively. The black dotted line represents linear regression (R2 = 0.25, P = 0.0004). (C) Paired data showing spine head volume for SP- vs SP+ spines. Each pair of plot represents average values for SP- vs SP+ spines for a given neuron. *P = 0.0031 (two-tailed Student t test). Data information: Data are represented as mean ± SEM.


( F ) Higher magnification of dendritic spines from neurons transfected with rescue RFP - SP ( magenta ) containing wild - type ( WT ) or mutated ( MUT ) 3 ' UTR and BFP - shRNA - SP ( grey ) and contacted by GFP - Syp + terminals ( green ) . Arrowheads indicate putative synaptic contacts . Scale bar : 5 μm . ( G ) Percentage of SP + spines in neurons expressing RFP - SP - 3 ' UTR - WT vs - MUT and depending on the apposition or not to a GFP - Syp + terminal ( SP - 3 ' UTR - WT : TetTx - , n = 220 , TetTx + , n = 26 ; SP - 3 ' UTR - MUT : TetTx - , n = 616 , TetTx + , n = 25 ; n indicates the number of spines from 5 - 7 pairs ) . ( H ) Spine head volume in same conditions as in ( G ) ( SP - 3 ' UTR - WT : TetTx - , n = 26 , TetTx + , n = 26 spines from 5 pairs , 4 slices ; SP - 3 ' UTR - MUT : TetTx - , n = 25 , TetTx + , n = 25 ; n indicates the number of spines from 7 pairs , 7 slices ) . * * * P < 0 . 001 , * P < 0 . 05 , ns , not significant ( Kruskall - Wallis test followed by Dunn ' s multiple test ) . ( I ) Cumulative probability distributions of spine head volumes for TetTx - and TetTx + spines from neurons expressing RFP - SP - 3 ' UTR - WT ( SP - WT , light gray and light green ) or RFP - SP - 3 ' UTR - MUT ( SP - MUT , dark gray and dark green ) . Data information : Data are represented as mean ± SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Higher magnification of dendritic spines from neurons transfected with rescue RFP-SP (magenta) containing wild-type (WT) or mutated (MUT) 3'UTR and BFP-shRNA-SP (grey) and contacted by GFP-Syp+ terminals (green). Arrowheads indicate putative synaptic contacts. Scale bar: 5 μm. (G) Percentage of SP+ spines in neurons expressing RFP-SP-3'UTR-WT vs -MUT and depending on the apposition or not to a GFP-Syp+ terminal (SP-3'UTR-WT: TetTx-, n = 220, TetTx+, n = 26; SP-3'UTR-MUT: TetTx-, n = 616, TetTx+, n = 25; n indicates the number of spines from 5-7 pairs). (H) Spine head volume in same conditions as in (G) (SP-3'UTR-WT: TetTx-, n = 26, TetTx+, n = 26 spines from 5 pairs, 4 slices; SP-3'UTR-MUT: TetTx-, n = 25, TetTx+, n = 25; n indicates the number of spines from 7 pairs, 7 slices). ***P < 0.001, *P < 0.05, ns, not significant (Kruskall-Wallis test followed by Dunn's multiple test). (I) Cumulative probability distributions of spine head volumes for TetTx- and TetTx+ spines from neurons expressing RFP-SP-3'UTR-WT (SP-WT, light gray and light green) or RFP-SP-3'UTR-MUT (SP-MUT, dark gray and dark green). Data information: Data are represented as mean ± SEM.


Colony formation assay of miRNA mimic transfected - Dgcr8 - / - ESCs . There were 17 miRNAs that decreased the colony forming ability , 15 miRNAs that did not affect colony formation and 8 miRNAs enhanced colony formation of ESCs . Representative pictures are shown . Full data are shown in Supplementary Fig S1 .

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

Colony formation assay of miRNA mimic transfected-Dgcr8-/- ESCs. There were 17 miRNAs that decreased the colony forming ability, 15 miRNAs that did not affect colony formation and 8 miRNAs enhanced colony formation of ESCs. Representative pictures are shown. Full data are shown in Supplementary Fig S1.


Alkaline phosphatase staining of ESCs after miRNA mimic transfection . There were 21 miRNAs that decreased the AP activity , 11 miRNAs that mildly affected the AP activity and 8 miRNAs boosted the AP activity of ESCs . Representative pictures are shown . Full data are shown in Supplementary Fig S2 .

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

Alkaline phosphatase staining of ESCs after miRNA mimic transfection. There were 21 miRNAs that decreased the AP activity, 11 miRNAs that mildly affected the AP activity and 8 miRNAs boosted the AP activity of ESCs. Representative pictures are shown. Full data are shown in Supplementary Fig S2.


Cell cycle distribution in representative miRNAs evoking G1 phase accumulation in miRNA mimic transfected Dgcr8 - / - ESCs . Error bars indicate s . d . ( n = 3 ) . Full data are shown in Supplementary Fig S3A , B .

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

Cell cycle distribution in representative miRNAs evoking G1 phase accumulation in miRNA mimic transfected Dgcr8-/- ESCs. Error bars indicate s. d. (n=3). Full data are shown in Supplementary Fig S3A, B.


Immunofluorescence staining of Oct4 in miRNA mimic transfected ESCs . 20 miRNAs decreased Oct4 expression and produced small and grossly differentiated cell colonies , 12 miRNAs mildly affected Oct4 expression , and 8 miRNAs improved Oct4 expression and yielded compact and undifferentiated cell colonies . Representative pictures are shown . Full data are shown in Supplementary Fig S3C .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Oct4 in miRNA mimic transfected ESCs. 20 miRNAs decreased Oct4 expression and produced small and grossly differentiated cell colonies, 12 miRNAs mildly affected Oct4 expression, and 8 miRNAs improved Oct4 expression and yielded compact and undifferentiated cell colonies. Representative pictures are shown. Full data are shown in Supplementary Fig S3C.


AP staining of V6 . 5 ESCs after mimic transfection ( Full data are shown in Supplementary Fig S4B ) .

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

AP staining of V6.5 ESCs after mimic transfection (Full data are shown in Supplementary Fig S4B).


B , C . Northern blot analysis of the expression of miR - 23a , miR - 27a and miR - 24 during EB formation ( B ) and RA induced differentiation of V6 . 5 ESCs ( C ) . The heatmap was representive of the results of quantitative analysis .

O O O O O 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

B,C. Northern blot analysis of the expression of miR-23a, miR-27a and miR-24 during EB formation (B) and RA induced differentiation of V6.5 ESCs (C). The heatmap was representive of the results of quantitative analysis.


D , E . The expression of primary miR - 23a ~ 27a ~ 24 - 2 transcript during EB formation ( D ) and RA induced differentiation ( E ) . Error bars indicate s . d . ( n = 3 ) .

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

D,E. The expression of primary miR-23a~27a~24-2 transcript during EB formation (D) and RA induced differentiation (E). Error bars indicate s.d. (n=3).


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


F . Q - PCR analysis of miR - 23a , miR - 27a and miR - 24 expression in ESCs ( JM8A3 ) and a variety of C57BL / 6J adult tissues . Data are shown as mean + s . d . ( n = 3 ) .

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

F. Q-PCR analysis of miR-23a, miR-27a and miR-24 expression in ESCs (JM8A3) and a variety of C57BL/6J adult tissues. Data are shown as mean + s.d. (n=3).


G . Relative protein levels of Oct4 , Sox2 and Nanog in miRNA mimic transfected Dgcr8 - / - ESCs and wild - type ESCs .

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

G. Relative protein levels of Oct4, Sox2 and Nanog in miRNA mimic transfected Dgcr8-/- ESCs and wild- type ESCs.


H , I . Oct4 , Nanog , Foxa2 , Gata2 and Nestin immunofluorescence staining of mimic transfected Dgcr8 - / - ( H ) and wild - type ESCs ( I ) .

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

H,I. Oct4, Nanog, Foxa2, Gata2 and Nestin immunofluorescence staining of mimic transfected Dgcr8-/- (H) and wild- type ESCs(I).


Western blot analysis of the expression of target genes in miR - 27a or miR - 24 over - expressed V6 . 5 ESCs . Candidate target genes whose expression was decreased were framed .

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

Western blot analysis of the expression of target genes in miR-27a or miR-24 over-expressed V6.5 ESCs. Candidate target genes whose expression was decreased were framed.


The relative luciferase activity of the reporter constructs co - transfected with scramble or miR - 27a and miR - 24 mimics . Error bars indicate s . d . ( n = 3 ) ; * indicates P - value < 0 . 05 . Candidate target genes whose luciferase activity was decreased were framed . PC group was positive reporter control which harbored reverse complementary sequence of miR - 27a or miR - 24 .

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

The relative luciferase activity of the reporter constructs co-transfected with scramble or miR-27a and miR-24 mimics. Error bars indicate s.d. (n=3); * indicates P-value < 0.05. Candidate target genes whose luciferase activity was decreased were framed. PC group was positive reporter control which harbored reverse complementary sequence of miR-27a or miR-24.


The relative luciferase activity of the wild - type 3 ′ UTR or mutated 3 ′ UTR reporter co - transfected with scramble or miR - 27a and miR - 24 mimics . Data are shown as mean + s . d . ( n = 3 ) ; * indicates P - value < 0 . 05 .

The relative luciferase activity of the wild-type 3′ UTR or mutated 3′ UTR reporter co-transfected with scramble or miR-27a and miR-24 mimics. Data are shown as mean + s.d. (n=3); * indicates P-value < 0.05.


F , G RT - PCR and qRT - PCR analysis of in vivo binding of the miRNAs with their targets . Error bars indicate s . d . ( n = 3 ) ; * indicates P - value < 0 . 05 ; * * indicates P - value < 0 . 01 ; * * * indicates P - value < 0 . 001 .

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

F,G RT-PCR and qRT-PCR analysis of in vivo binding of the miRNAs with their targets. Error bars indicate s.d. (n=3); * indicates P-value < 0.05; ** indicates P-value < 0.01; *** indicates P-value < 0.001.


H . Western blot analysis of c - Myc expression in siRNA treated V6 . 5 ESCs .

H. Western blot analysis of c-Myc expression in siRNA treated V6.5 ESCs.


I . Q - PCR analysis of primary - 23a ~ 27a ~ 24 - 2 , miR - 23a , miR - 27a and miR - 24 expression in c - Myc knocked down ESCs . Error bars indicate s . d . ( n = 3 ) ; * indicates P - value < 0 . 05 ; * * indicates P - value < 0 . 01 ; * * * indicates P - value < 0 . 001 .

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

I. Q-PCR analysis of primary-23a~27a~24-2, miR-23a, miR-27a and miR-24 expression in c-Myc knocked down ESCs. Error bars indicate s.d. (n=3); * indicates P-value < 0.05; ** indicates P-value < 0.01; *** indicates P-value < 0.001.


K . ChIP - PCR and ChIP - q - PCR analysis of c - Myc binding to the putative sites in the promoter . Error bars indicate s . d . ( n = 3 ) ; * * * indicates P - value < 0 . 001 .

K. ChIP-PCR and ChIP-q-PCR analysis of c-Myc binding to the putative sites in the promoter. Error bars indicate s.d. (n=3); *** indicates P-value < 0.001.


L . Relative luciferase activity of the wild - type and - 15 binding site mutated promoter in NIH3T3 cells co - transfected with c - Myc or the blank vector ( NC ) . Error bars indicate s . d . ( n = 3 ) ; * indicates P - value < 0 . 05 .

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

L. Relative luciferase activity of the wild-type and -15 binding site mutated promoter in NIH3T3 cells co-transfected with c-Myc or the blank vector (NC). Error bars indicate s.d. (n=3); * indicates P-value < 0.05.


M . Relative luciferase activity of wild - type and - 15 binding site mutated promoter in V6 . 5 ESCs . Error bars indicate s . d . ( n = 3 ) ; * indicates P - value < 0 . 05 .

M. Relative luciferase activity of wild-type and -15 binding site mutated promoter in V6.5 ESCs. Error bars indicate s.d. (n=3); * indicates P-value < 0.05.


Validation of the effect of miRNA inhibition in MEFs by q - PCR . Error bars indicate s . d . ( n = 3 ) .

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

Validation of the effect of miRNA inhibition in MEFs by q-PCR. Error bars indicate s.d. (n=3).


AP staining of the iPSC colonies in reprogramming with OSK combined with scramble control , inhibitors of miR - 23a , miR - 27a , miR - 24 and let - 7c respectively .

AP staining of the iPSC colonies in reprogramming with OSK combined with scramble control, inhibitors of miR-23a, miR-27a, miR-24 and let-7c respectively.


Fold increase in Oct4 - GFP - positive cell colonies in the above cells . Data are shown as mean + s . d . ( n = 5 ) ; * * indicates P - value < 0 . 01 ; * * * indicates P - value < 0 . 001 .

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

Fold increase in Oct4-GFP-positive cell colonies in the above cells. Data are shown as mean + s.d. (n=5); ** indicates P-value < 0.01; *** indicates P-value < 0.001.


Morphology of typical Oct4 - GFP - positive iPSC colonies that were reprogrammed by OSK combined with miRNA inhibitors .

Morphology of typical Oct4-GFP-positive iPSC colonies that were reprogrammed by OSK combined with miRNA inhibitors.


Detection of endogenous and exogenous Oct4 , Sox2 , Klf4 and Nanog expression in iPSCs , ESCs and MEFs by RT - PCR .

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

Detection of endogenous and exogenous Oct4, Sox2, Klf4 and Nanog expression in iPSCs, ESCs and MEFs by RT-PCR.


Immunofluorescence staining of Oct4 , Nanog and SSEA1 of the iPSC colonies .

Immunofluorescence staining of Oct4, Nanog and SSEA1 of the iPSC colonies.


Q - PCR analysis of the expression of self - renewal ( Oct4 , Sox2 , Nanog ) and differentiation ( Cdx2 , Foxa2 , Brachyury , Hand1 , Nestin , FGF5 ) relative markers in EBs derived from the iPSCs . Error bars indicate s . d . ( n = 3 ) .

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Q-PCR analysis of the expression of self-renewal (Oct4, Sox2, Nanog) and differentiation (Cdx2, Foxa2, Brachyury, Hand1, Nestin, FGF5) relative markers in EBs derived from the iPSCs. Error bars indicate s.d. (n=3).


Hematoxylin and eosin staining of teratoma derived from the iPSC colonies . Neuronal rosette ( Ectoderm ) , Cartilage ( Mesoderm ) and respiratory epithelium ( Endoderm ) are marked with black arrows .

Hematoxylin and eosin staining of teratoma derived from the iPSC colonies. Neuronal rosette (Ectoderm), Cartilage (Mesoderm) and respiratory epithelium (Endoderm) are marked with black arrows.


Genomic PCR of the specific fragments spanning miR - 23 ~ 27 ~ 24 clusters . WT indicate wild - type ESCs ; DKO indicate miR - 23a ~ 27a ~ 24 - 2 - / - , miR - 23b ~ 27b ~ 24 - 1 - / - ESCs ; KO indicate miR - 23a ~ 27a ~ 24 - 2 - / - ESCs .

Genomic PCR of the specific fragments spanning miR-23~27~24 clusters. WT indicate wild-type ESCs; DKO indicate miR-23a~27a~24-2-/-, miR-23b~27b~24-1-/- ESCs; KO indicate miR-23a~27a~24-2-/- ESCs.


Real - time PCR analysis of mature miR - 23a / b , miR - 27a / b and miR - 24 expression in wild - type , DKO and KO ESCs . Data are shown as mean + s . d . ( n = 3 ) .

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Real-time PCR analysis of mature miR-23a/b, miR-27a/b and miR-24 expression in wild-type, DKO and KO ESCs. Data are shown as mean + s.d. (n=3).


AP staining of wild - type and miR - 23 ~ 27 ~ 24 cluster knockout ESCs .

AP staining of wild-type and miR-23~27~24 cluster knockout ESCs.


Real - time PCR analysis of Oct4 , Sox2 and Nanog expression in wild - type and miR - 23 ~ 27 ~ 24 cluster knockout ESCs . Data are shown as mean + s . d . ( n = 3 ) .

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Real-time PCR analysis of Oct4, Sox2 and Nanog expression in wild-type and miR-23~27~24 cluster knockout ESCs. Data are shown as mean + s.d. (n=3).


Oct4 , Sox2 and Nanog immunofluorescence staining of wild - type and the knockout ESCs .

Oct4, Sox2 and Nanog immunofluorescence staining of wild-type and the knockout ESCs.


Real - time PCR analysis of Oct4 and several lineage differentiation markers expression during EB formation of wild - type and knockout ESCs . Data are shown as mean ± s . d . ( n = 3 ) .

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Real-time PCR analysis of Oct4 and several lineage differentiation markers expression during EB formation of wild-type and knockout ESCs. Data are shown as mean ± s.d. (n=3).


Beating EB incidence during cardiac differentiation of wild - type and knockout ESCs . Data are shown as mean + s . d . ( n = 3 ) . * indicates P - value < 0 . 05 ; * * indicates P - value < 0 . 01 ; * * * indicates P - value < 0 . 001 .

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Beating EB incidence during cardiac differentiation of wild-type and knockout ESCs. Data are shown as mean + s.d. (n=3). * indicates P-value < 0.05; ** indicates P-value < 0.01; *** indicates P-value < 0.001.


Real - time PCR analysis of several cardiac markers expression during cardiac differentiation of wild - type and knockout ESCs . Data are shown as mean ± s . d . ( n = 3 ) .

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Real-time PCR analysis of several cardiac markers expression during cardiac differentiation of wild-type and knockout ESCs. Data are shown as mean ± s.d. (n=3).


Actinin , ANP and Troponin Iimmunofluorescence staining of differentiation cultures derived from wild - type and knockout ESCs .

Actinin, ANP and Troponin Iimmunofluorescence staining of differentiation cultures derived from wild-type and knockout ESCs.


Volumes of teratomas at different days after injection . Data are shown as mean + s . d . ( n = 4 ) . * indicates P - value < 0 . 05 ; * * indicates P - value < 0 . 01 ; * * * indicates P - value < 0 . 001 .

Volumes of teratomas at different days after injection. Data are shown as mean + s.d. (n=4). * indicates P-value < 0.05; ** indicates P-value < 0.01; *** indicates P-value < 0.001.


Weight of teratomas derived from wild - type and knockout ESCs . Data are shown as mean ± s . d . * * indicates P - value < 0 . 01 .

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Weight of teratomas derived from wild-type and knockout ESCs. Data are shown as mean ± s.d. ** indicates P-value < 0.01.


Oct4 immunohistochemistry staining of teratomas derived from wild - type and knockout ESCs . Representative Oct4 positive regions are shown .

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Oct4 immunohistochemistry staining of teratomas derived from wild-type and knockout ESCs. Representative Oct4 positive regions are shown.


Hematoxylin and eosin staining of teratomas derived from wild - type and knockout ESCs . Ectoderm , Mesoderm and Endoderm are marked with different color arrows .

Hematoxylin and eosin staining of teratomas derived from wild-type and knockout ESCs. Ectoderm, Mesoderm and Endoderm are marked with different color arrows.


Orthotopic MPNST xenografts at passages 1 ( OT P1 ) and 4 ( OT P4 ) were histopathologically similar to their corresponding primary MPNST ( PT ) in hematoxylin - eosin staining of paraffin - embedded tumor sections from patients MPNST - NF1 - 001 and MPNST - SP - 002 . Main panels show a general view of the tumors at low magnification ( 40 × ) ; inset pictures were taken at higher magnification ( 400 × ) .

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Orthotopic MPNST xenografts at passages 1 (OT P1) and 4 (OT P4) were histopathologically similar to their corresponding primary MPNST (PT) in hematoxylin-eosin staining of paraffin-embedded tumor sections from patients MPNST-NF1-001 and MPNST-SP-002. Main panels show a general view of the tumors at low magnification (40×); inset pictures were taken at higher magnification (400×).


Orthotopic xenograft and primary MPNSTs exhibited similar immunohistochemical features . A representative immunostained section of vimentin , CD34 , S100 , and Ki - 67 is shown for primary tumors ( PT ) and orthotopic tumors ( OT ) from patientsMPNST - NF1 - 001 and MPNST - SP - 002 . Positive antibody signals are shown in brown , and the hematoxylin counterstain in blue . Main panels show pictures at high magnification ( 400 × ) ; inset pictures show mitotic cells present in these tumors .

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Orthotopic xenograft and primary MPNSTs exhibited similar immunohistochemical features. A representative immunostained section of vimentin, CD34, S100, and Ki-67 is shown for primary tumors (PT) and orthotopic tumors (OT) from patientsMPNST-NF1-001 and MPNST-SP-002. Positive antibody signals are shown in brown, and the hematoxylin counterstain in blue. Main panels show pictures at high magnification (400×); inset pictures show mitotic cells present in these tumors.


Stromal elements of the primary tumors were labeled with anti - humanCD34 but not anti - mouseCD34 ; patient - derived xenografts are labeled with anti - mouseCD34 only and no anti - human marker . Representative sections ( at 40 × and 400 × magnification ) of the primary tumors ( PT ) and the orthoxenograft tumors at passages 1 ( OT P1 ) and 4 ( OT P4 ) were labeled with anti - humanCD34 ( H ) and anti - mouseCD34 ( M ) .

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Stromal elements of the primary tumors were labeled with anti-humanCD34 but not anti-mouseCD34; patient-derived xenografts are labeled with anti-mouseCD34 only and no anti-human marker. Representative sections (at 40× and 400× magnification) of the primary tumors (PT) and the orthoxenograft tumors at passages 1 (OT P1) and 4 (OT P4) were labeled with anti-humanCD34 (H) and anti-mouseCD34 (M).


Sanger sequencing of the germline NF1 mutation c . 350T > A present in the patient that developed two different tumors ( MPNST - NF1 - 001 and MPNST - NF1 - 002 ) . Sequencing results from normal tissue , primary tumor ( PT ) , and orthotopic xenograft tumors ( OT ) are shown . The sequence of the NF1 region revealed WT NF1 alleles in primary tumor samples , and not in the corresponding derived orthoxenografts , indicating probable loss of the human stromal cells .

Sanger sequencing of the germline NF1 mutation c.350T>A present in the patient that developed two different tumors (MPNST-NF1-001 and MPNST-NF1-002). Sequencing results from normal tissue, primary tumor (PT), and orthotopic xenograft tumors (OT) are shown. The sequence of the NF1 region revealed WT NF1 alleles in primary tumor samples, and not in the corresponding derived orthoxenografts, indicating probable loss of the human stromal cells.


SNP array of primary tumor ( PT ) and orthotopic xenograft passages 1 ( OT P1 ) and 4 ( OT P4 ) for MPNST - NF1 - 001 and MPNST - NF1 - 002 tumors . Results correspond to chromosome 17 . Images show the B - allele frequency ( BAF ) for the different samples as scatter plots and the copy number callings below them represented by thick horizontal lines : 2n regions are shown in gray , gained regions in orange , lost regions in green , and LOH regions are represented in blue . The vertical red line indicates the location of the NF1 locus . In addition , the percentage of tumor versus stromal cells for each sample is represented in a pie chart ( blue , stromal cells ; red , tumor cells ) .

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SNP array of primary tumor (PT) and orthotopic xenograft passages 1 (OT P1) and 4 (OT P4) for MPNST-NF1-001 and MPNST-NF1-002 tumors. Results correspond to chromosome 17. Images show the B-allele frequency (BAF) for the different samples as scatter plots and the copy number callings below them represented by thick horizontal lines: 2n regions are shown in gray, gained regions in orange, lost regions in green, and LOH regions are represented in blue. The vertical red line indicates the location of the NF1 locus. In addition, the percentage of tumor versus stromal cells for each sample is represented in a pie chart (blue, stromal cells; red, tumor cells).


Tumor growth effects of treatment with doxorubicin , sorafenib , rapamycin and combinations thereof in the five MPNST xenograft models . Results are plotted as an average of the log2 ratio of tumor volume at different days relative to the initial value . Statistically significant differences are shown as * P < 0 . 05 and * * P < 0 . 001 versus control group by the Bonferroni test .

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Tumor growth effects of treatment with doxorubicin, sorafenib, rapamycin and combinations thereof in the five MPNST xenograft models. Results are plotted as an average of the log2 ratio of tumor volume at different days relative to the initial value. Statistically significant differences are shown as *P < 0.05 and **P < 0.001 versus control group by the Bonferroni test.


For long - term studies , a subgroup of treated mice ( n = 3 - 5 mice / group ) was kept alive for a maximum period of 4 months and sacrificed over time when relapsed tumor masses grew as large solid masses ( usually 1 , 500 - 2 , 000 mm3 ) . The graph illustrates differences in the time delay ( in days ) of relapsed tumor masses for the different treatments .

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For long-term studies, a subgroup of treated mice (n = 3-5 mice/group) was kept alive for a maximum period of 4 months and sacrificed over time when relapsed tumor masses grew as large solid masses (usually 1,500-2,000 mm3). The graph illustrates differences in the time delay (in days) of relapsed tumor masses for the different treatments.


D Distributions of pairwise Ka / Ks ( nonsynonymous to synonymous nucleotide substitution rates ) ratios between PIWI duplicates in arthropods ( n = 17 in Drosophila and n = 37 in others , Mann - Whitney U test , * P < 0 . 05 ) . The center line of the boxplots represents median value , the bounds of the box represent 75th and 25th percentile , and the whiskers represent maximum and minimum value .

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D Distributions of pairwise Ka/Ks (nonsynonymous to synonymous nucleotide substitution rates) ratios between PIWI duplicates in arthropods (n = 17 in Drosophila and n = 37 in others, Mann-Whitney U test, *P<0.05). The center line of the boxplots represents median value, the bounds of the box represent 75th and 25th percentile, and the whiskers represent maximum and minimum value.


A PIWI genes are present in the locust brain . Rp49 served as a loading control ( n = 4 biological replicates ) . Data information : data are presented as mean ± SEM .

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A PIWI genes are present in the locust brain. Rp49 served as a loading control (n=4 biological replicates). Data information: data are presented as mean ± SEM.


C The fluorescence signals of PIWI proteins were determined in locust brains . Hochest33342 was used to label cell nuclei in locust brains . The scale bars represent 20 µm .

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C The fluorescence signals of PIWI proteins were determined in locust brains. Hochest33342 was used to label cell nuclei in locust brains. The scale bars represent 20 µm.


G Food intake by dsPiwi1 - treated locusts and dsGFP - treated control locusts within 1 h ( n = 5 biological replicates ) , * * * P < 0 . 001 ( Student ' s t - test ) . Data information : All assays were performed on fourth - instar locusts .

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G Food intake by dsPiwi1-treated locusts and dsGFP-treated control locusts within 1 h (n=5 biological replicates) , ***P<0.001 (Student's t-test). Data information: All assays were performed on fourth-instar locusts.


C Representative images of Nile red staining of lipid droplets in the fat bodies of dsPiwi1 - treated locusts and dsGFP - treated control locusts ( n = 5 biological replicates ) . The scale bars represent 50 µm . D Quantification of lipid droplet staining in dsPiwi1 - treated locusts and dsGFP - treated control locusts ( n = 5 biological replicates ) . E Data information : All data are presented as the mean ± SEM ( Student ' s t - test , * * P < 0 . 01 , * * * P < 0 . 001 , ns : not significant ) . All assays were performed on four - day - old fourth - instar locusts .

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C Representative images of Nile red staining of lipid droplets in the fat bodies of dsPiwi1-treated locusts and dsGFP-treated control locusts (n=5 biological replicates). The scale bars represent 50 µm. D Quantification of lipid droplet staining in dsPiwi1-treated locusts and dsGFP-treated control locusts (n=5 biological replicates). E Data information: All data are presented as the mean ± SEM (Student's t-test, **P<0.01, ***P<0.001, ns: not significant). All assays were performed on four-day-old fourth-instar locusts.


E , F Measurements of glycogen ( n = 5 biological replicates ) and TAGs ( n = 10 biological replicates ) in fat bodies . Data information : All data are presented as the mean ± SEM ( Student ' s t - test , * * P < 0 . 01 , * * * P < 0 . 001 , ns : not significant ) . All assays were performed on four - day - old fourth - instar locusts .

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E, F Measurements of glycogen (n=5 biological replicates) and TAGs (n=10 biological replicates) in fat bodies. Data information: All data are presented as the mean ± SEM (Student's t-test, **P<0.01, ***P<0.001, ns: not significant). All assays were performed on four-day-old fourth-instar locusts.


B Food intake within 1 h following treatment with candidate genes ( dsNPF1 - treated , n = 4 biological replicates ; dsCG10621 - treated , n = 5 biological replicates ; dsMFS3 - treated , n = 5 biological replicates ) , the data are shown as the mean ± SEM ( Student ' s t - test , * * P < 0 . 01 ) .

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B Food intake within 1 h following treatment with candidate genes (dsNPF1-treated, n=4 biological replicates; dsCG10621-treated, n=5 biological replicates; dsMFS3-treated, n=5 biological replicates), the data are shown as the mean ± SEM (Student's t-test, **P<0.01).


D , E Representative images ( D ) and quantification ( E ) of Nile red staining of fat body lipid droplets in dsNPF1 - treated locusts and dsGFP - treated controls ( n = 5 biological replicates ) . The scale bars represent 50 µm , the data are shown as the mean ± SEM ( Student ' s t - test , * * P < 0 . 01 ) .

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D, E Representative images (D) and quantification (E) of Nile red staining of fat body lipid droplets in dsNPF1-treated locusts and dsGFP-treated controls (n=5 biological replicates). The scale bars represent 50 µm, the data are shown as the mean ± SEM (Student's t-test, **P<0.01).


G Food intake within 1 h in the food instake assays ( n = 5 biological replicates ) , the data are shown as the mean ± SEM ( Student ' s t - test , * P < 0 . 05 , * * * P < 0 . 001 ) . H Food intake within 2 h in the food instake assays ( n = 5 biological replicates ) . Five locusts were included in each replicate , the data are shown as the mean ± SEM ( Student ' s t - test , * P < 0 . 05 , * * P < 0 . 01 ) .

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G Food intake within 1 h in the food instake assays (n=5 biological replicates), the data are shown as the mean ± SEM (Student's t-test, *P<0.05, ***P<0.001) . H Food intake within 2 h in the food instake assays (n=5 biological replicates). Five locusts were included in each replicate, the data are shown as the mean ± SEM (Student's t-test, *P<0.05, **P<0.01).


B mRNA levels of differentially expressed piRNAs located within the flanking region of the NPF1 gene in dsPiwi1 - treated locusts and dsGFP - treated control locusts ( n = 5 biological replicates ) . Data information : The qPCR data are shown as the mean ± SEM ( Student ' s t - test , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

B mRNA levels of differentially expressed piRNAs located within the flanking region of the NPF1 gene in dsPiwi1-treated locusts and dsGFP-treated control locusts (n=5 biological replicates). Data information: The qPCR data are shown as the mean ± SEM (Student's t-test, *P<0.05, **P<0.01, ***P<0.001).


C The mRNA expression of NPF1 was measured by qPCR after a mixture of inhibitors or mimics of piRs - 3 - I2 and piRs - 3 - I3 was injected ( n = 5 biological replicates ) . Data information : The qPCR data are shown as the mean ± SEM ( Student ' s t - test , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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C The mRNA expression of NPF1 was measured by qPCR after a mixture of inhibitors or mimics of piRs-3-I2 and piRs-3-I3 was injected (n=5 biological replicates). Data information: The qPCR data are shown as the mean ± SEM (Student's t-test, *P<0.05, **P<0.01, ***P<0.001).


E Body weight measurement of locusts administered inhibitors ( NC , n = 25 locusts ; Inhibitors - piRs - 3 - I2 + I3 , n = 23 locusts ) and mimics ( NC , n = 37 locusts ; Inhibitors - piRs - 3 - I2 + I3 , n = 39 locusts ) of piRs - 3 - I2 and piRs - 3 - I3 . The center line of the boxplots represents median value , the bounds of the box represent 75th and 25th percentile , and the whiskers represent maximum and minimum value .

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E Body weight measurement of locusts administered inhibitors (NC, n=25 locusts; Inhibitors- piRs-3-I2+I3, n=23 locusts) and mimics (NC, n=37 locusts; Inhibitors-piRs-3-I2+I3, n=39 locusts) of piRs-3-I2 and piRs-3-I3. The center line of the boxplots represents median value, the bounds of the box represent 75th and 25th percentile, and the whiskers represent maximum and minimum value.


F RNA - binding protein immunoprecipitation assay of piRs - 3 - I2 and piRs - 3 - I3 in Piwi1 immunoprecipitates from brain tissue extracts ( n = 6 biological replicates ) . Normal mouse IgG was used as a negative control .

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F RNA-binding protein immunoprecipitation assay of piRs-3-I2 and piRs-3-I3 in Piwi1 immunoprecipitates from brain tissue extracts (n=6 biological replicates). Normal mouse IgG was used as a negative control.


A U6 RNA was used as a nuclear RNA control ( relative value set to 1 in the nuclear fraction ) , and glycine tRNA was used as the cytoplasmic RNA control ( relative value set to 1 in the cytoplasmic fraction ) ( n = 4 biological replicates ) , U6 and tRNA - Gly was used as an endogenous control .

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A U6 RNA was used as a nuclear RNA control (relative value set to 1 in the nuclear fraction), and glycine tRNA was used as the cytoplasmic RNA control (relative value set to 1 in the cytoplasmic fraction) (n=4 biological replicates), U6 and tRNA-Gly was used as an endogenous control.


B Relative expressions of piRs - 3 - I2 and piRs - 3 - I3 determined by qPCR and high - throughput sequencing in the nucleus ( n = 3 biological replicates ) . The brain tissues were homogenized and then divided into two parts of equal volume ; one was prepared for high - throughput sequencing and the other was used for piRNA quantification in nuclear and cytoplasmic fractions after subcellular RNA fractionation . The relative piRNA expression in nucleus was determined by multiplying the relative nuclear piRNA fraction estimate by qPCR quantification by the normalized count of piRNA in high - throughput sequencing libraries . Data information : The qPCR data are shown as the mean ± SEM ( Student ' s t - test , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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B Relative expressions of piRs-3-I2 and piRs-3-I3 determined by qPCR and high-throughput sequencing in the nucleus (n=3 biological replicates). The brain tissues were homogenized and then divided into two parts of equal volume; one was prepared for high-throughput sequencing and the other was used for piRNA quantification in nuclear and cytoplasmic fractions after subcellular RNA fractionation. The relative piRNA expression in nucleus was determined by multiplying the relative nuclear piRNA fraction estimate by qPCR quantification by the normalized count of piRNA in high-throughput sequencing libraries. Data information: The qPCR data are shown as the mean ± SEM (Student's t-test, *P<0.05, **P<0.01, ***P<0.001).


C Expression level of pre - NPF1 in the nucleus after a mixture of inhibitors ( piRs - 3 - I2 and piRs - 3 - I3 ) was injected ( n = 4 biological replicates ) . D qPCR expression analyses of pre - NPF1 . The precursor mRNA expression level was measured by qPCR after the dsPiwi1 treatments ( n = 6 biological replicates ) . E Data information : The qPCR data are shown as the mean ± SEM ( Student ' s t - test , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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C Expression level of pre-NPF1 in the nucleus after a mixture of inhibitors (piRs-3-I2 and piRs-3-I3) was injected (n=4 biological replicates). D qPCR expression analyses of pre-NPF1. The precursor mRNA expression level was measured by qPCR after the dsPiwi1 treatments (n=6 biological replicates). E Data information: The qPCR data are shown as the mean ± SEM (Student's t-test, *P<0.05, **P<0.01, ***P<0.001).


F Analysis was performed to amplify pre - NPF1 and piRs - 3 - I3 in hnRNP F / H immunoprecipitates from extracts of brain tissue treated with piRs - 3 - I3 mimics compared to the negative controls ( pre - NPF1 n = 3 biological replicates ; piRs - 3 - I3 n = 4 biological replicates ) . G Analysis was performed to amplify pre - NPF1 in U2AF65 immunoprecipitates from extracts of brain tissue treated with piRs - 3 - I3 mimics compared to the negative controls ( pre - NPF1 n = 3 biological replicates ) .

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F Analysis was performed to amplify pre-NPF1 and piRs-3-I3 in hnRNP F/H immunoprecipitates from extracts of brain tissue treated with piRs-3-I3 mimics compared to the negative controls (pre-NPF1 n=3 biological replicates; piRs-3-I3 n=4 biological replicates). G Analysis was performed to amplify pre-NPF1 in U2AF65 immunoprecipitates from extracts of brain tissue treated with piRs-3-I3 mimics compared to the negative controls (pre-NPF1 n=3 biological replicates). H


J Expression level of the mature NPF1 gene and pre - NPF1 in 293T cells transfected with pre - NPF1 and mutated pre - NPF1 ( n = 5 biological replicates ) . NC means negative control . The center line of the boxplots represents median value , the bounds of the box represent 75th and 25th percentile , and the whiskers represent maximum and minimum value . Data information : The qPCR data are shown as the mean ± SEM ( Student ' s t - test , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Expression level of the mature NPF1 gene and pre-NPF1 in 293T cells transfected with pre-NPF1 and mutated pre-NPF1 (n=5 biological replicates). NC means negative control. The center line of the boxplots represents median value, the bounds of the box represent 75th and 25th percentile, and the whiskers represent maximum and minimum value. Data information: The qPCR data are shown as the mean ± SEM (Student's t-test, *P<0.05, **P<0.01, ***P<0.001).


NE activates mTORC2 in vitro via cAMP , PI3K and Epac1 . ( A ) Immunoblot analysis of BAT cells stimulated with norepinephrine ( NE ) for the indicated proteins .

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

NE activates mTORC2 in vitro via cAMP, PI3K and Epac1. (A) Immunoblot analysis of BAT cells stimulated with norepinephrine (NE) for the indicated proteins.


( B ) Immunoblot analysis of BAT cells stimulated with NE for 5 minutes in the presence of Rapamycin ( Rapa ) , Torin or Wortmannin ( Wrtm ) for the indicated proteins .

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

(B) Immunoblot analysis of BAT cells stimulated with NE for 5 minutes in the presence of Rapamycin (Rapa), Torin or Wortmannin (Wrtm) for the indicated proteins.


( C ) Immunoblot analysis of BAT cells stimulated with 8 - Br - cAMP for 5 minutes in the presence of Rapa , Torin or Wrtm for the indicated proteins .

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

(C) Immunoblot analysis of BAT cells stimulated with 8-Br-cAMP for 5 minutes in the presence of Rapa, Torin or Wrtm for the indicated proteins.


( D ) Immunoblot analysis of BAT cells stimulated with NE for 5 minutes in the presence of Wrtm , H89 or ESI - 09 for the indicated proteins .

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

(D) Immunoblot analysis of BAT cells stimulated with NE for 5 minutes in the presence of Wrtm, H89 or ESI-09 for the indicated proteins.


( E ) Immunoblot analysis of BAT cells stimulated with 8 - Br - cAMP for 5 minutes in the presence of Wrtm , H89 or ESI - 09 for the indicated proteins . All experiments were performed in triplicates , a representative replicate is presented .

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

(E) Immunoblot analysis of BAT cells stimulated with 8-Br-cAMP for 5 minutes in the presence of Wrtm, H89 or ESI-09 for the indicated proteins. All experiments were performed in triplicates, a representative replicate is presented.


NE and cold activate mTORC2 in vivo . ( A ) Immunoblot analysis of BAT from control mice treated with either norepinephrine ( NE ) or vehicle for 30 minutes for the indicated proteins ( n = 3 / group ) .

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

NE and cold activate mTORC2 in vivo. (A) Immunoblot analysis of BAT from control mice treated with either norepinephrine (NE) or vehicle for 30 minutes for the indicated proteins (n=3/group).


( B ) Immunoblot analysis of BAT from AdRiKO and control mice treated with either norepinephrine ( NE ) or vehicle for 30 minutes for the indicated proteins ( n = 3 / group ) .

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

(B) Immunoblot analysis of BAT from AdRiKO and control mice treated with either norepinephrine (NE) or vehicle for 30 minutes for the indicated proteins (n=3/group).


( C ) Immunoblot analysis of BAT from AdRiKO and control mice housed at either 22 ° C or 4 ° C for 2h for the indicated proteins ( n = 6 / group , each lane represents a mix of 3 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 O O O O

(C) Immunoblot analysis of BAT from AdRiKO and control mice housed at either 22°C or 4°C for 2h for the indicated proteins (n=6/group, each lane represents a mix of 3 mice).


( D ) Body temperature of AdRiKO and control mice housed at 22 ° C ( n = 11 ( control ) , n = 9 ( AdRiKO ) ) .

O O O O O O O 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 AdRiKO and control mice housed at 22°C (n=11 (control), n=9 (AdRiKO)).


( E ) Body temperature of AdRiKO and control mice housed at 30 ° C for 2 weeks ( n = 8 / 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

(E) Body temperature of AdRiKO and control mice housed at 30°C for 2 weeks (n=8/group).


( F ) Body temperature loss upon cold exposure of AdRiKO and control mice ( n = 20 ( control ) , n = 17 ( AdRiKO ) ) . Data represent mean ± SEM . Statistically significant differences between AdRiKO and control mice were determined with unpaired Student ' s t - test and are indicated with asterisks ( * = p < 0 . 05 ; * * = p < 0 . 01 , * * * = p < 0 . 001 ) . Statistically significant differences between temperatures are indicated with a number sign ( # = p < 0 . 05 ; # # = p < 0 . 01 ; # # # = p < 0 . 001 ) . The exact p - value for each significant difference can be found in Appendix Table S2 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Body temperature loss upon cold exposure of AdRiKO and control mice (n=20 (control), n=17 (AdRiKO)). Data represent mean ± SEM. Statistically significant differences between AdRiKO and control mice were determined with unpaired Student's t-test and are indicated with asterisks (*=p<0.05; **=p<0.01, ***=p<0.001). Statistically significant differences between temperatures are indicated with a number sign (#=p<0.05; ##=p<0.01; ###=p<0.001). The exact p-value for each significant difference can be found in Appendix Table S2.


mTORC2 in adipose tissue is not required for cold - induced lipid droplet mobilization . ( A ) Representative H & amp ; amp ; E staining of sWAT sections from AdRiKO and control mice ( n = 5 / 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

mTORC2 in adipose tissue is not required for cold-induced lipid droplet mobilization. (A) Representative H&amp;E staining of sWAT sections from AdRiKO and control mice (n=5/group).


( B ) Non - esterified fatty acids ( NEFAs ) in plasma of AdRiKO and control mice ( n = 6 / group ) .

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

(B) Non-esterified fatty acids (NEFAs) in plasma of AdRiKO and control mice (n=6/group).


( C ) Glycerol in plasma of AdRiKO and control mice ( n = 6 / group ) .

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

(C) Glycerol in plasma of AdRiKO and control mice (n=6/group).


( D ) Representative H & amp ; amp ; E staining of BAT sections from AdRiKO and control mice ( n = 5 / 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

(D) Representative H&amp;E staining of BAT sections from AdRiKO and control mice (n=5/group).


( E ) Triglycerides ( TGs ) in BAT of AdRiKO and control mice housed at 22 ° C or 4 ° C for 8h ( n = 6 / group ) .

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

(E) Triglycerides (TGs) in BAT of AdRiKO and control mice housed at 22°C or 4°C for 8h (n=6/group).


( F ) NEFAs in BAT of AdRiKO and control mice ( n = 6 / group ) . Data represent mean ± SEM . Statistically significant differences between AdRiKO and control mice were determined with unpaired Student ' s t - test and are indicated with asterisks ( * = p < 0 . 05 ; * * = p < 0 . 01 , * * * = p < 0 . 001 ) . Statistically significant differences between temperatures were determined with unpaired Student ' s t - test and are indicated with a number sign ( # = p < 0 . 05 ; # # = p < 0 . 01 ; # # # = p < 0 . 001 ) . The exact p - value for each significant difference can be found in Appendix Table S2 .

O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) NEFAs in BAT of AdRiKO and control mice (n=6/group). Data represent mean ± SEM. Statistically significant differences between AdRiKO and control mice were determined with unpaired Student's t-test and are indicated with asterisks (*=p<0.05; **=p<0.01, ***=p<0.001). Statistically significant differences between temperatures were determined with unpaired Student's t-test and are indicated with a number sign (#=p<0.05; ##=p<0.01; ###=p<0.001). The exact p-value for each significant difference can be found in Appendix Table S2.


mTORC2 in adipose tissue is not required for cold - induced mitochondrial uncoupling and β - oxidation . ( A ) mRNA levels of the indicated genes in BAT of AdRiKO and control mice housed at 22 ° C or at 4 ° C for 8h ( n = 6 / 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

mTORC2 in adipose tissue is not required for cold-induced mitochondrial uncoupling and β-oxidation. (A) mRNA levels of the indicated genes in BAT of AdRiKO and control mice housed at 22°C or at 4°C for 8h (n=6/group).


( B ) Immunoblot analysis of BAT from AdRiKO and control mice housed at 22 ° C or at 4 ° C for 8h for the indicated proteins ( n = 6 / group , each lane represents a mix of 3 mice ) .

(B) Immunoblot analysis of BAT from AdRiKO and control mice housed at 22°C or at 4°C for 8h for the indicated proteins (n=6/group, each lane represents a mix of 3 mice).


( C ) mRNA levels of the indicated genes in BAT of AdRiKO and control mice housed at 22 ° C or at 4 ° C for 8h ( n = 6 / group , each lane represents a mix of 3 mice ) .

(C) mRNA levels of the indicated genes in BAT of AdRiKO and control mice housed at 22°C or at 4°C for 8h (n=6/group, each lane represents a mix of 3 mice).


( D ) Immunoblot analysis of BAT from AdRiKO and control mice housed at 22 ° C or at 4 ° C for 8h for the indicated proteins ( n = 6 / group , each lane represents a mix of 3 mice ) .

(D) Immunoblot analysis of BAT from AdRiKO and control mice housed at 22°C or at 4°C for 8h for the indicated proteins (n=6/group, each lane represents a mix of 3 mice).


( E ) Mitochondrial DNA content of BAT from AdRiKO and control mice housed at 22 ° C or at 4 ° C for 8h ( n = 6 / 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

(E) Mitochondrial DNA content of BAT from AdRiKO and control mice housed at 22°C or at 4°C for 8h (n=6/group).


( F ) Representative electronmicrographs of BAT from AdRiKO and control mice housed at 22 ° C or at 4 ° C for 4h ( n = 3 / 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

(F) Representative electronmicrographs of BAT from AdRiKO and control mice housed at 22°C or at 4°C for 4h (n=3/group).


( G ) Oxygen consumption rate ( OCR ) of BAT explants from AdRiKO and control mice housed at 22 ° C or at 4 ° C for 4h ( n = 7 / group ) .

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

(G) Oxygen consumption rate (OCR) of BAT explants from AdRiKO and control mice housed at 22°C or at 4°C for 4h (n=7/group).


H ) Maximal respiration ( VO2 max ) of AdRiKO and control mice housed at 22 ° C or at 4 ° C for 8h ( n = 9 ( control 22 ° C ) , n = 7 ( AdRiKO 22 ° C ) , n = 8 ( control 4 ° C ) , n = 8 ( AdRiKO 4 ° C ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Maximal respiration (VO2 max) of AdRiKO and control mice housed at 22°C or at 4°C for 8h (n=9 (control 22°C), n=7 (AdRiKO 22°C), n=8 (control 4°C), n=8 (AdRiKO 4°C).


( I ) Respiration ( VO2 ) of AdRiKO and control mice upon cold exposure ( n = 8 / group ) . Data represent mean ± SEM . Statistically significant differences between AdRiKO and control mice were determined with unpaired Student ' s t - test and are indicated with asterisks ( * = p < 0 . 05 ; * * = p < 0 . 01 , * * * = p < 0 . 001 ) . Statistically significant differences between temperatures were determined with unpaired Student ' s t - test and are indicated with a number sign ( # = p < 0 . 05 ; # # = p < 0 . 01 ; # # # = p < 0 . 001 ) . The exact p - value for each significant difference can be found in Appendix Table S2 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Respiration (VO2) of AdRiKO and control mice upon cold exposure (n=8/group). Data represent mean ± SEM. Statistically significant differences between AdRiKO and control mice were determined with unpaired Student's t-test and are indicated with asterisks (*=p<0.05; **=p<0.01, ***=p<0.001). Statistically significant differences between temperatures were determined with unpaired Student's t-test and are indicated with a number sign (#=p<0.05; ##=p<0.01; ###=p<0.001). The exact p-value for each significant difference can be found in Appendix Table S2.


mTORC2 in adipose tissue is required for cold - induced glucose uptake and glycolysis ( A ) 2 - Deoxyglucose - 6 - phosphate ( 2DG6P ) accumulation in BAT of AdRiKO and control mice housed at 22 ° C or at 4 ° C for 4h ( n = 6 / group ) . (

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 I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-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

mTORC2 in adipose tissue is required for cold-induced glucose uptake and glycolysis (A) 2-Deoxyglucose-6-phosphate (2DG6P) accumulation in BAT of AdRiKO and control mice housed at 22°C or at 4°C for 4h (n=6/group). (


( B ) Extracellular acidification rate ( ECAR ) of BAT explants from AdRiKO and control mice housed at 22 ° C or at 4 ° C for 4h ( n = 7 / 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

(B) Extracellular acidification rate (ECAR) of BAT explants from AdRiKO and control mice housed at 22°C or at 4°C for 4h (n=7/group).


( C ) Immunoblot analysis of BAT from AdRiKO and control mice housed at 22 ° C or at 4 ° C for 8h for the indicated proteins ( n = 6 / group , each lane represents a mix of 3 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 O O O O

(C) Immunoblot analysis of BAT from AdRiKO and control mice housed at 22°C or at 4°C for 8h for the indicated proteins (n=6/group, each lane represents a mix of 3 mice).


( D ) Immunoblot analysis of isolated plasma membranes from BAT of AdRiKO and control mice housed at 22 ° C or at 4 ° C for 8h for the indicated proteins ( n = 6 / group , each lane represents a mix of 3 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 O O O O O O O O

(D) Immunoblot analysis of isolated plasma membranes from BAT of AdRiKO and control mice housed at 22°C or at 4°C for 8h for the indicated proteins (n=6/group, each lane represents a mix of 3 mice).


( E ) Immunoblot analysis of mitochondrial and cytosolic fractions from BAT of AdRiKO and control mice housed at 22 ° C or at 4 ° C for 4h for the indicated proteins ( n = 6 / group , each lane represents a mix of 3 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 O O O O O O O O O

(E) Immunoblot analysis of mitochondrial and cytosolic fractions from BAT of AdRiKO and control mice housed at 22°C or at 4°C for 4h for the indicated proteins (n=6/group, each lane represents a mix of 3 mice).


( F ) Cytosolic hexokinase activity in BAT of AdRiKO and control mice housed at 22 ° C or at 4 ° C for 4h ( n = 5 ( control 22 ° C ) , n = 5 ( AdRiKO 22 ° C ) , n = 7 ( control 4 ° C ) , n = 7 AdRiKO 4 ° C ) ) .

(F) Cytosolic hexokinase activity in BAT of AdRiKO and control mice housed at 22°C or at 4°C for 4h (n=5 (control 22°C), n=5 (AdRiKO 22°C), n=7 (control 4°C), n=7 AdRiKO 4°C)).


( G ) Mitochondrial hexokinase activity in BAT of AdRiKO and control mice housed at 22 ° C or at 4 ° C for 4h ( n = 5 ( control 22 ° C ) , n = 5 ( AdRiKO 22 ° C ) , n = 7 ( control 4 ° C ) , n = 7 AdRiKO 4 ° C ) ) . Data represent mean ± SEM . Statistically significant differences between AdRiKO and control mice were determined with unpaired Student ' s t - test and are indicated with asterisks ( * = p < 0 . 05 ; * * = p < 0 . 01 , * * * = p < 0 . 001 ) . Statistically significant differences between temperatures were determined with unpaired Student ' s t - test and are indicated with a number sign ( # = p < 0 . 05 ; # # = p < 0 . 01 ; # # # = p < 0 . 001 ) . The exact p - value for each significant difference can be found in Appendix Table S2 .

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

(G) Mitochondrial hexokinase activity in BAT of AdRiKO and control mice housed at 22°C or at 4°C for 4h (n=5 (control 22°C), n=5 (AdRiKO 22°C), n=7 (control 4°C), n=7 AdRiKO 4°C)). Data represent mean ± SEM. Statistically significant differences between AdRiKO and control mice were determined with unpaired Student's t-test and are indicated with asterisks (*=p<0.05; **=p<0.01, ***=p<0.001). Statistically significant differences between temperatures were determined with unpaired Student's t-test and are indicated with a number sign (#=p<0.05; ##=p<0.01; ###=p<0.001). The exact p-value for each significant difference can be found in Appendix Table S2.


Restoration of glucoseuptake or Akt signaling suppresses the thermogenic defect in AdRiKO mice . ( A ) HKII mRNA expression level in BAT of AdRiKO and control mice infected with either AAV9 - HKII or AAV9 - empty ( n = 8 / 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

Restoration of glucoseuptake or Akt signaling suppresses the thermogenic defect in AdRiKO mice. (A) HKII mRNA expression level in BAT of AdRiKO and control mice infected with either AAV9-HKII or AAV9-empty (n=8/group).


( B ) Cold - induced 2 - Deoxyglucose - 6 - phosphate ( 2DG6P ) accumulation in BAT of AdRiKO and control mice infected with either AAV9 - HKII or AAV9 - empty housed at 4 ° C for 4h ( n = 8 / group ) .

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

(B) Cold-induced 2-Deoxyglucose-6-phosphate (2DG6P) accumulation in BAT of AdRiKO and control mice infected with either AAV9-HKII or AAV9-empty housed at 4°C for 4h (n=8/group).


( C ) Body temperature of AdRiKO and control mice infected with either AAV9 - HKII or AAV9 - empty housed at 22 ° C ( n = 8 / group ) .

(C) Body temperature of AdRiKO and control mice infected with either AAV9-HKII or AAV9-empty housed at 22°C (n=8/group).


( D ) Body temperature upon cold exposure of AdRiKO and control mice infected with either AAV9 - HKII or AAV9 - empty . The left panel represents body temperature after each hour of cold exposure while the right panel represents body temperature as a bar graph for the 3h cold exposure time point ( n = 8 / group ) . a = significant difference between AdRiKO and control mice infected with AAV9 - empty , b = significant difference between AdRiKO and control mice infected with AAV9 - HKII , d = significant difference between AdRiKO mice infected with AAV9 - empty and AAV9 - HKII .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 upon cold exposure of AdRiKO and control mice infected with either AAV9-HKII or AAV9-empty. The left panel represents body temperature after each hour of cold exposure while the right panel represents body temperature as a bar graph for the 3h cold exposure time point (n=8/group). a=significant difference between AdRiKO and control mice infected with AAV9-empty, b= significant difference between AdRiKO and control mice infected with AAV9-HKII, d= significant difference between AdRiKO mice infected with AAV9-empty and AAV9-HKII.


( E ) Immunoblot analysis of BAT from AdRiKO and control mice infected with either AAV8 - Akt2S474D or AAV8 - empty ( n = 6 / group , each lane represents a mix of 3 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

(E) Immunoblot analysis of BAT from AdRiKO and control mice infected with either AAV8-Akt2S474D or AAV8-empty (n=6/group, each lane represents a mix of 3 mice).


( F ) Body temperature of AdRiKO and control mice infected with either AAV8 - Akt2S474D or AAV8 - empty housed at 22 ° C ( n = 11 / group ) .

(F) Body temperature of AdRiKO and control mice infected with either AAV8-Akt2S474D or AAV8-empty housed at 22°C (n=11/group).


( G ) Body temperature upon cold exposure of AdRiKO and control mice infected with either AAV8 - Akt2S474D or AAV8 - empty . The left panel represents body temperature after each hour of cold exposure while the right panel represents body temperature as a bar graph for the 3h cold exposure time point ( n = 11 / group ) . a = significant difference between AdRiKO and control mice infected with AAV8 - empty , b = significant difference between AdRiKO and control mice infected with AAV8 - Akt2S474D , d = significant difference between AdRiKO mice infected with AAV8 - empty and AAV8 - Akt2S474D .

(G) Body temperature upon cold exposure of AdRiKO and control mice infected with either AAV8-Akt2S474D or AAV8-empty. The left panel represents body temperature after each hour of cold exposure while the right panel represents body temperature as a bar graph for the 3h cold exposure time point (n=11/group). a=significant difference between AdRiKO and control mice infected with AAV8-empty, b= significant difference between AdRiKO and control mice infected with AAV8-Akt2S474D, d= significant difference between AdRiKO mice infected with AAV8-empty and AAV8-Akt2S474D.


( H ) Cold - induced 2 - Deoxyglucose - 6 - phosphate ( 2DG6P ) accumulation in BAT of AdRiKO and control mice infected with either AAV8 - Akt2S474D or AAV8 - empty housed at 4 ° C for 4h ( n = 7 ( control AAV8 - null ) , n = 6 ( AdRiKO AAV8 - null ) , n = 6 ( control AAV8 - AktS474D ) , n = 6 ( AdRiKO AAV8 - AktS474D ) ) . Data represent mean ± SEM . Statistically significant differences between AdRiKO and control mice were determined with unpaired Student ' s t - test and are indicated with asterisks ( * = p < 0 . 05 ; * * = p < 0 . 01 , * * * = p < 0 . 001 ) . Statistically significant differences between viruses were determined with unpaired Student ' s t - test and are indicated with a number sign ( # = p < 0 . 05 ; # # = p < 0 . 01 ; # # # = p < 0 . 001 ) . The exact p - value for each significant difference can be found in Appendix Table S2 .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cold-induced 2-Deoxyglucose-6-phosphate (2DG6P) accumulation in BAT of AdRiKO and control mice infected with either AAV8-Akt2S474D or AAV8-empty housed at 4°C for 4h (n=7 (control AAV8-null), n=6 (AdRiKO AAV8-null), n=6 (control AAV8-AktS474D), n=6 (AdRiKO AAV8-AktS474D)). Data represent mean ± SEM. Statistically significant differences between AdRiKO and control mice were determined with unpaired Student's t-test and are indicated with asterisks (*=p<0.05; **=p<0.01, ***=p<0.001). Statistically significant differences between viruses were determined with unpaired Student's t-test and are indicated with a number sign (#=p<0.05; ##=p<0.01; ###=p<0.001). The exact p-value for each significant difference can be found in Appendix Table S2.


Blistered samples at P1 . ( B ) Hematoxylin and eosin ( H & E , top ) and alkaline phosphatase ( AP , bottom ) staining . Hair follicles ( HFs ) remaining in the dermis ( indicated by arrows ) . Scale bar : 500 μm . ( C ) α6 Integrin ( ITGA6 , indicated by arrowheads ) and type IV collagen ( COL4 , arrows ) labeling ( left ) . Laminin 332 ( L332 , arrows ) staining ( right ) . Scale bar : 100 μm . ( D ) Ultrastructural findings of blistered skin ( left image : blister roof , right image : blister bottom ) . Hemidesmosomes ( white arrowheads ) and lamina densa ( arrows ) are indicated . 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 I-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Blistered samples at P1. (B) Hematoxylin and eosin (H&E, top) and alkaline phosphatase (AP, bottom) staining. Hair follicles (HFs) remaining in the dermis (indicated by arrows). Scale bar: 500 μm. (C) α6 Integrin (ITGA6, indicated by arrowheads) and type IV collagen (COL4, arrows) labeling (left). Laminin 332 (L332, arrows) staining (right). Scale bar: 100 μm. (D) Ultrastructural findings of blistered skin (left image: blister roof, right image: blister bottom) . Hemidesmosomes (white arrowheads) and lamina densa (arrows) are indicated. Scale bar: 1 μm.


Keratin 14 ( K14 ) and keratin 10 ( K10 ) staining of the nonlesional ( intact ) and lesional ( blistered ) skin at P2 ( upper images and inlets : sections , lower images : whole - mount imaging ) . HFs are indicated by arrows in the whole - mount images . Scale bar : 30 μm .

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Keratin 14 (K14) and keratin 10 (K10) staining of the nonlesional (intact) and lesional (blistered) skin at P2 (upper images and inlets: sections, lower images: whole-mount imaging). HFs are indicated by arrows in the whole-mount images. Scale bar: 30 μm.


H & E ( left ) , PCK ( middle ) , and LOR staining ( right ) at P4 . Scale bar : 200 μm .

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H&E (left), PCK (middle), and LOR staining (right) at P4. Scale bar: 200 μm.


Scatter plots of differentially expressed genes in the regenerated epidermis . The red dots represent upregulated genes , and the blue dots represent downregulated genes . The gray dotted lines indicate | logFC | > 1 .

Scatter plots of differentially expressed genes in the regenerated epidermis. The red dots represent upregulated genes, and the blue dots represent downregulated genes. The gray dotted lines indicate |logFC|>1.


Hair canals in the regenerated ( lesional ) and nonlesional epidermis at P4 ( indicated by asterisks ) . Scale bar : 300 μm .

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Hair canals in the regenerated (lesional) and nonlesional epidermis at P4 (indicated by asterisks). Scale bar: 300 μm.


BrdU labeling of blistered samples at P2 . Scale bar : 100 μm . BrdU - positive cells are indicated by arrows . Blisters are indicated by stars . ( Bottom ) Quantification of BrdU - positive cells in the epidermis ( left ) and HFs ( right ) ( n = 4 biological replicates ) . The data are shown as the mean ± SE . * 0 . 01 test , followed by Tukey ' s test . NS , no significance .

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

BrdU labeling of blistered samples at P2. Scale bar: 100 μm. BrdU-positive cells are indicated by arrows. Blisters are indicated by stars. (Bottom) Quantification of BrdU-positive cells in the epidermis (left) and HFs (right) (n=4 biological replicates). The data are shown as the mean ± SE. *0.01 test, followed by Tukey's test. NS, no significance.


α5 integrin ( ITGA5 ) labeling at P2 ( left image : section , right image : whole - mount ) . Scale bar : 100 μm . Blister edges ( epidermal tongue ) and HFs are indicated by arrowheads and arrows , respectively . Blisters are indicated by stars .

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α5 integrin (ITGA5) labeling at P2 (left image: section, right image: whole-mount). Scale bar: 100 μm. Blister edges (epidermal tongue) and HFs are indicated by arrowheads and arrows, respectively. Blisters are indicated by stars.


Sections of K14CreER : H2B - mCherry mice at P4 . Scale bar : 100 μm . ( Bottom ) Quantification of mCherry - positive cells ( n = 3 ) . . The data from individual mice are connected by lines . Student ' s t - test . NS , no significance . Whole - mount imaging of K14CreER : R26R - confetti samples at P4 . Scale bar : 200 μm . Sections of Lrig1CreER : H2B - mCherry mouse skin at P4 . Scale bar : 100 μm . ( Bottom ) Quantification of mCherry - positive cells ( n = 3 ) . The data from individual mice are connected by lines . * 0 . 01 test . Whole - mount imaging of Lrig1CreER : R26R - confetti mouse samples at P4 . Scale bar : 200 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Sections of K14CreER:H2B-mCherry mice at P4. Scale bar: 100 μm. (Bottom) Quantification of mCherry-positive cells (n=3).. The data from individual mice are connected by lines. Student's t-test. NS, no significance. Whole-mount imaging of K14CreER:R26R-confetti samples at P4. Scale bar: 200 μm. Sections of Lrig1CreER:H2B-mCherry mouse skin at P4. Scale bar: 100 μm. (Bottom) Quantification of mCherry-positive cells (n=3). The data from individual mice are connected by lines. *0.01 test. Whole-mount imaging of Lrig1CreER:R26R-confetti mouse samples at P4. Scale bar: 200 μm.


H & E ( P2 , left ) , ITGA6 ( P2 , middle ) and LOR staining ( P4 , right ) of Col17a1 - / - mice ( top ) and littermate controls ( bottom ) . The regenerated epidermis is indicated by arrowheads . Scale bar : 200 μm .

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H&E (P2, left), ITGA6 (P2, middle) and LOR staining (P4, right) of Col17a1-/- mice (top) and littermate controls (bottom) . The regenerated epidermis is indicated by arrowheads. Scale bar: 200 μm.


BrdU labeling of Col17a1 - / - skin at P2 . Scale bar : 100 μm . ( Bottom ) Quantification of BrdU - positive cells in the epidermis surrounding blisters ( n = 3 ( control ) and 4 ( Col17a1 - / - ) biological replicates ) . The data are shown as the mean ± SE . Student ' s t - test . NS , no significance .

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

BrdU labeling of Col17a1-/- skin at P2. Scale bar: 100 μm. (Bottom) Quantification of BrdU-positive cells in the epidermis surrounding blisters (n= 3 (control) and 4 (Col17a1-/-) biological replicates). The data are shown as the mean ± SE. Student's t-test. NS, no significance.


Lineage tracing of K14CreER : R26R - mCherry : Col17a1 - / - at P4 . Scale bar : 100 μm .

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Lineage tracing of K14CreER:R26R-mCherry:Col17a1-/- at P4. Scale bar: 100 μm.


K10 / K14 ( low magnification , left ) and K14 ( high magnification , middle and right ) labeling of Col7a1 - / - mouse ( bottom ) and littermate control ( top ) blistered skin at P2 . Scale bar : 30 μm . Quantification of BrdU - positive cells per μm HF length ( n = 55 HFs from three control and 143 HFs from four Col7a1 - / - mice ) . The data are shown as violin plots . Student ' s t - test . NS , no significance . Length of the major axis of keratinocytes in the regenerated epidermis ( n = 244 ( control , L ) , and 132 ( Col7a1 - / - , L ) cells from four mice , respectively ) and in the surrounding intact epidermis ( basal cells ; n = 200 ( control , NL ) , 299 ( Col7a1 - / - , NL ) , from four mice , respectively ) . NL : nonlesional area . L : lesional area . The data are shown as violin plots . * * * * p < 0 . 0001 , one - way ANOVA test , followed by Tukey ' s test . NS , no significance .

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

K10/K14 (low magnification, left) and K14 (high magnification, middle and right) labeling of Col7a1-/- mouse (bottom) and littermate control (top) blistered skin at P2. Scale bar: 30 μm. Quantification of BrdU-positive cells per μm HF length (n=55 HFs from three control and 143 HFs from four Col7a1-/- mice). The data are shown as violin plots. Student's t-test. NS, no significance. Length of the major axis of keratinocytes in the regenerated epidermis (n=244 (control, L), and 132 (Col7a1-/-, L) cells from four mice, respectively) and in the surrounding intact epidermis (basal cells; n=200 (control, NL), 299 (Col7a1-/-, NL), from four mice, respectively). NL: nonlesional area. L: lesional area. The data are shown as violin plots. ****p<0.0001, one-way ANOVA test, followed by Tukey's test. NS, no significance.


K10 / K14 ( left ) and K14 ( high magnification , right ) labeling of WT blistered skin treated with CaCl2 ( middle and bottom ) or PBS ( top ) at P2 . Scale bar : 30 μm . Quantification of BrdU - positive cells per μm HF length ( n = 83 ( PBS ) , 95 ( 1 . 8 mM CaCl2 ) , and 97 ( 9 . 0 mM CaCl2 ) HFs from four mice ) . One - way ANOVA test , followed by Tukey ' s test . NS , no significance . Length of the major axis of keratinocytes in the regenerated epidermis ( n = 433 ( PBS , L ) , 451 ( 1 . 8 mM CaCl2 , L ) , and 425 ( 9 . 0 mM CaCl2 , L ) cells from four mice ) and in the surrounding intact epidermis ( basal cells ; n = 311 ( PBS , NL ) , 279 ( 1 . 8 mM CaCl2 , NL ) , 302 ( 9 . 0 mM CaCl2 , NL ) cells from four mice ) . NL : nonlesional area . L : lesional area . The data are shown as violin plots . * * * * p < 0 . 0001 , one - way ANOVA test , followed by Tukey ' s test . NS , no significance .

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 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 O O O O O O 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 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-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

K10/K14 (left) and K14 (high magnification, right) labeling of WT blistered skin treated with CaCl2 (middle and bottom) or PBS (top) at P2. Scale bar: 30 μm. Quantification of BrdU-positive cells per μm HF length (n=83 (PBS), 95 (1.8 mM CaCl2), and 97 (9.0 mM CaCl2) HFs from four mice). One-way ANOVA test, followed by Tukey's test. NS, no significance. Length of the major axis of keratinocytes in the regenerated epidermis (n=433 (PBS, L), 451 (1.8 mM CaCl2, L), and 425 (9.0 mM CaCl2, L) cells from four mice) and in the surrounding intact epidermis (basal cells; n=311 (PBS, NL), 279 (1.8 mM CaCl2, NL), 302 (9.0 mM CaCl2, NL) cells from four mice). NL: nonlesional area. L: lesional area. The data are shown as violin plots. ****p<0.0001, one-way ANOVA test, followed by Tukey's test. NS, no significance.


( D ) The indicated amounts of recombinant wild - type and mutant PPM1H or PPM1J ( with a His - Sumo N - terminal tag , expressed in E . coli ) were incubated in vitro with 2 . 5 µg pThr72 phosphorylated Rab8a ( left ) or pThr73 phosphorylated Rab10 ( right ) for 20 min in the presence of 10 mM MgCl2 in 40 mM HEPES pH 7 . 5 buffer . Reactions were terminated by addition of SDS Sample Buffer and analyzed by Phos - tag gel electrophoresis that separates phosphorylated ( slow migrating ) and dephosphorylated Rabs . The gel was stained with Instant Blue Coomassie . D288A is a substrate - trapping ( inactive ) variant of PPM1H and was used as a control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The indicated amounts of recombinant wild-type and mutant PPM1H or PPM1J (with a His-Sumo N-terminal tag, expressed in E. coli) were incubated in vitro with 2.5 µg pThr72 phosphorylated Rab8a (left) or pThr73 phosphorylated Rab10 (right) for 20 min in the presence of 10 mM MgCl2 in 40 mM HEPES pH 7.5 buffer. Reactions were terminated by addition of SDS Sample Buffer and analyzed by Phos-tag gel electrophoresis that separates phosphorylated (slow migrating) and dephosphorylated Rabs. The gel was stained with Instant Blue Coomassie. D288A is a substrate-trapping (inactive) variant of PPM1H and was used as a control.


( D ) Kinetics of phosphate hydrolysis . 25 nM recombinant wild - type PPM1H was incubated with increasing concentrations of pThr72 phosphorylated Rab8a ( GTPγS or GDP ) as described in Materials and Methods . Initial velocity ( V0 ) was calculated by dividing the concentration of released phosphatase ( μM ) by time ( min ) and plotted against substrate concentration for pThr72 phosphorylated Rab8a [ GTP bound conformation ] ( blue ) and pThr72 phosphorylated Rab8a [ GDP bound conformation ] ( red ) . The experiments were repeated twice , and both data points are shown in curves . Line fittings for the left and middle panel were performed using the mean of the two values . Kinetic constants ( Kcat , Vmax , Km ) were obtained using GraphPad software , and their uncertainties ( ± ) correspond to the SE of mean .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Kinetics of phosphate hydrolysis. 25 nM recombinant wild-type PPM1H was incubated with increasing concentrations of pThr72 phosphorylated Rab8a (GTPγS or GDP) as described in Materials and Methods. Initial velocity (V0) was calculated by dividing the concentration of released phosphatase (μM) by time (min) and plotted against substrate concentration for pThr72 phosphorylated Rab8a[GTP bound conformation] (blue) and pThr72 phosphorylated Rab8a[GDP bound conformation] (red). The experiments were repeated twice, and both data points are shown in curves. Line fittings for the left and middle panel were performed using the mean of the two values. Kinetic constants (Kcat, Vmax, Km) were obtained using GraphPad software, and their uncertainties (±) correspond to the SE of mean.


( E ) Kinetic analysis of PPM1H as in ( D ) using 50 nM PPM1H and phosphopeptide substrates , as described in Materials and Methods . Initial velocity ( V0 ) was calculated by dividing the concentration of phosphatase ( μM ) by time ( min ) and plotted against substrate concentration for pThr72 Rab8a phospho - peptide ( blue ) and Rab10 pThr73 phospho - peptide ( red ) . Each experiment was performed twice ( individual data points shown ) .

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

(E) Kinetic analysis of PPM1H as in (D) using 50 nM PPM1H and phosphopeptide substrates, as described in Materials and Methods. Initial velocity (V0) was calculated by dividing the concentration of phosphatase (μM) by time (min) and plotted against substrate concentration for pThr72 Rab8a phospho-peptide (blue) and Rab10 pThr73 phospho-peptide (red). Each experiment was performed twice (individual data points shown).


( F ) Side by side comparison of the catalytic activity against protein and peptide by in vitro malachite green time course analysis . 50 nM recombinant wild - type PPM1H was incubated with 16 μM Rab8a GTPγS pThr72 phosphorylated protein ( blue ) or Rab8a pThr72 phosphorylated peptide ( red ) for indicated times and analysed as described in Materials and Methods . The experiments were repeated 3 times . The error bars represent SE of mean of the technical 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 O O O O O O O O O O O O O O O O O O O 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) Side by side comparison of the catalytic activity against protein and peptide by in vitro malachite green time course analysis. 50 nM recombinant wild-type PPM1H was incubated with 16 μM Rab8a GTPγS pThr72 phosphorylated protein (blue) or Rab8a pThr72 phosphorylated peptide (red) for indicated times and analysed as described in Materials and Methods. The experiments were repeated 3 times. The error bars represent SE of mean of the technical replicates.


( C ) His6 - SUMO - tagged full - length variants of PPM1H were used for catalytic assays ( left panel ) . 3 μg of recombinant wild - type or indicated mutant PPM1H proteins were resolved on 4 - 12 % Bis - Tris gradient gel and stained with Instant Blue Coomassie . Right , mass photometry histogram for 40nM His6 - SUMO - PPM1H WT ( blue ) and His6 - SUMO - PPM1H 2Glu ( brown ) , where WT is 130kDa ( ± 15 . 8 kDa , with 1004 single molecules counted ) and 2Glu is 83kDa ( ± 13 . 9 kDa , with 4117 single molecules counted ) . The calculated molecular weight of the fusion protein is approximately 68 . 5 kDa .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) His6-SUMO-tagged full-length variants of PPM1H were used for catalytic assays (left panel). 3 μg of recombinant wild-type or indicated mutant PPM1H proteins were resolved on 4-12% Bis-Tris gradient gel and stained with Instant Blue Coomassie. Right, mass photometry histogram for 40nM His6-SUMO-PPM1H WT (blue) and His6-SUMO-PPM1H 2Glu (brown), where WT is 130kDa (±15.8 kDa, with 1004 single molecules counted) and 2Glu is 83kDa (±13.9 kDa, with 4117 single molecules counted). The calculated molecular weight of the fusion protein is approximately 68.5 kDa.


( D ) In vitro malachite green assay time course of recombinant His6 - SUMO - tagged PPM1H wild - type ( blue ) , 2Glu ( red ) , or D288A ( grey ) against 16 μM pThr72 phosphorylated Rab8a protein ( GTPγS , left ) , 32 μM pThr72 phosphorylated Rab8a peptide ( middle ) , or 32 μM pThr73 phosphorylated Rab10 peptide ( right ) . The experiments were repeated 4 times . The error bars represent SE of mean of the technical 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 O O O O O O O O O O O O O O O O O O O O O O O O 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) In vitro malachite green assay time course of recombinant His6-SUMO-tagged PPM1H wild-type (blue), 2Glu (red), or D288A (grey) against 16 μM pThr72 phosphorylated Rab8a protein (GTPγS, left), 32 μM pThr72 phosphorylated Rab8a peptide (middle), or 32 μM pThr73 phosphorylated Rab10 peptide (right). The experiments were repeated 4 times. The error bars represent SE of mean of the technical replicates.