A - D Comparison of chymotrypsin - like activity from female ( n = 9 ) and male ( n = 10 ) mice in young ( 3 - 5 months old ) or older ( 10 - 15 months old , female ( n = 6 ) and male ( n = 7 ) ) groups . Each point indicates a single mouse . A ) Tissues that did not show significant decline , B ) Tissues that show significant decline in females only , C ) Tissues that show significant decline in males only or D ) in both males and females . Each central line and error bar indicate mean + / - SEM of a biological replicates ( n = 9 young females , n = 6 older females , n = 10 young males , n = 7 older males ) . P values obtained by unpaired two - tailed students t - test comparing young and older samples are indicated .

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A-D Comparison of chymotrypsin-like activity from female (n=9) and male (n=10) mice in young (3-5 months old) or older (10-15 months old, female (n=6) and male (n=7)) groups. Each point indicates a single mouse. A) Tissues that did not show significant decline, B) Tissues that show significant decline in females only, C) Tissues that show significant decline in males only or D) in both males and females. Each central line and error bar indicate mean +/- SEM of a biological replicates (n=9 young females, n=6 older females, n=10 young males, n=7 older males). P values obtained by unpaired two-tailed students t-test comparing young and older samples are indicated.


B ) Western blot analysis of Ub - K48 proteins in the soluble fractions of intestine lysates from mice plus or minus heat shock . C ) Quantification of panel B . Each central line and error bar indicate mean + / - SEM of a biological replicates ( n = 3 biological replicates per group ) . Data information : * indicates p < 0 . 05 , * * indicates p < 0 . 005 , and * * * indicates p < 0 . 0005 by unpaired two tailed students t - test comparing male and female samples or samples with or without heat shock .

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B) Western blot analysis of Ub-K48 proteins in the soluble fractions of intestine lysates from mice plus or minus heat shock. C) Quantification of panel B. Each central line and error bar indicate mean +/- SEM of a biological replicates (n=3 biological replicates per group). Data information: * indicates p<0.05, ** indicates p<0.005, and *** indicates p<0.0005 by unpaired two tailed students t-test comparing male and female samples or samples with or without heat shock.


D ) Western blot analysis of Ub - K48 proteins in the insoluble fraction of intestine lysates from mice plus or minus heat shock . E ) Quantification of D . Each central line and error bar indicate mean + / - SEM of a biological replicates ( n = 3 biological replicates per group ) Data information : * indicates p < 0 . 05 , * * indicates p < 0 . 005 , and * * * indicates p < 0 . 0005 by unpaired two tailed students t - test comparing male and female samples or samples with or without heat shock .

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 O

D) Western blot analysis of Ub-K48 proteins in the insoluble fraction of intestine lysates from mice plus or minus heat shock. E) Quantification of D. Each central line and error bar indicate mean +/- SEM of a biological replicates (n=3 biological replicates per group) Data information: * indicates p<0.05, ** indicates p<0.005, and *** indicates p<0.0005 by unpaired two tailed students t-test comparing male and female samples or samples with or without heat shock.


F ) Western blot analysis of Ub - K48 proteins in the soluble fractions of spinal cord lysates from mice plus or minus heat shock . G ) Quantification of F . Each central line and error bar indicate mean + / - SEM of a biological replicates ( n = 3 biological replicates per group ) Data information : * indicates p < 0 . 05 , * * indicates p < 0 . 005 , and * * * indicates p < 0 . 0005 by unpaired two tailed students t - test comparing male and female samples or samples with or without heat shock .

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F) Western blot analysis of Ub-K48 proteins in the soluble fractions of spinal cord lysates from mice plus or minus heat shock. G) Quantification of F. Each central line and error bar indicate mean +/- SEM of a biological replicates (n=3 biological replicates per group) Data information: * indicates p<0.05, ** indicates p<0.005, and *** indicates p<0.0005 by unpaired two tailed students t-test comparing male and female samples or samples with or without heat shock.


H ) Western blot analysis of Ub - K48 proteins in the insoluble fraction of spinal cord lysates from mice plus or minus heat shock . I ) Quantification of H . Each central line and error bar indicate mean + / - SEM of a biological replicates ( n = 3 biological replicates per group ) Data information : * indicates p < 0 . 05 , * * indicates p < 0 . 005 , and * * * indicates p < 0 . 0005 by unpaired two tailed students t - test comparing male and female samples or samples with or without heat shock .

H) Western blot analysis of Ub-K48 proteins in the insoluble fraction of spinal cord lysates from mice plus or minus heat shock. I) Quantification of H. Each central line and error bar indicate mean +/- SEM of a biological replicates (n=3 biological replicates per group) Data information: * indicates p<0.05, ** indicates p<0.005, and *** indicates p<0.0005 by unpaired two tailed students t-test comparing male and female samples or samples with or without heat shock.


K ) Western blot of Ub - K48 proteins in the insoluble fraction of protein lysates of SOD1 - G93A mouse spinal cord plus or minus heat shock . L ) Quantification of panel K . Each central line and error bar indicate mean + / - SEM of a biological replicates ( n = 5 biological replicates per group ) Data information : * indicates p < 0 . 05 , * * indicates p < 0 . 005 , and * * * indicates p < 0 . 0005 by unpaired two tailed students t - test comparing male and female samples or samples with or without heat shock .

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 O O O O O O O

K) Western blot of Ub-K48 proteins in the insoluble fraction of protein lysates of SOD1-G93A mouse spinal cord plus or minus heat shock. L) Quantification of panel K. Each central line and error bar indicate mean +/- SEM of a biological replicates (n=5 biological replicates per group) Data information: * indicates p<0.05, ** indicates p<0.005, and *** indicates p<0.0005 by unpaired two tailed students t-test comparing male and female samples or samples with or without heat shock.


M ) Chymotrypsin - like proteasome activity in spinal cord lysates from SOD1 - G93A mice . Each central line and error bar indicate mean + / - SEM of a biological replicates ( n = 5 biological replicates per group ) . Bordered points indicate biological replicates and un - bordered points indicate technical replicates . Data information : * indicates p < 0 . 05 , * * indicates p < 0 . 005 , and * * * indicates p < 0 . 0005 by unpaired two tailed students t - test comparing male and female samples or samples with or without heat shock .

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

M) Chymotrypsin-like proteasome activity in spinal cord lysates from SOD1-G93A mice. Each central line and error bar indicate mean +/- SEM of a biological replicates (n=5 biological replicates per group). Bordered points indicate biological replicates and un-bordered points indicate technical replicates. Data information: * indicates p<0.05, ** indicates p<0.005, and *** indicates p<0.0005 by unpaired two tailed students t-test comparing male and female samples or samples with or without heat shock.


N ) Filter trap assay of protein lysates from the insoluble fraction of SOD1 - G93A spinal cord lysates plus or minus heat shock . O ) Quantification of panel N . Each central line and error bar indicate mean + / - SEM of a biological replicates ( n = 5 biological replicates per group ) Data information : * indicates p < 0 . 05 , * * indicates p < 0 . 005 , and * * * indicates p < 0 . 0005 by unpaired two tailed students t - test comparing male and female samples or samples with or without heat shock .

N) Filter trap assay of protein lysates from the insoluble fraction of SOD1-G93A spinal cord lysates plus or minus heat shock. O) Quantification of panel N. Each central line and error bar indicate mean +/- SEM of a biological replicates (n=5 biological replicates per group) Data information: * indicates p<0.05, ** indicates p<0.005, and *** indicates p<0.0005 by unpaired two tailed students t-test comparing male and female samples or samples with or without heat shock.


P ) Representative transmission electron micrographs of protein aggregates in the insoluble fraction of SOD1 - G93A spinal cord lysates plus or minus heat shock . Scale bar equals 600nm . Q ) Quantification of P . ( n = 9 technical replicates per group ) Data information : * indicates p < 0 . 05 , * * indicates p < 0 . 005 , and * * * indicates p < 0 . 0005 by unpaired two tailed students t - test comparing male and female samples or samples with or without heat shock .

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

P) Representative transmission electron micrographs of protein aggregates in the insoluble fraction of SOD1-G93A spinal cord lysates plus or minus heat shock. Scale bar equals 600nm. Q) Quantification of P. (n=9 technical replicates per group) Data information: * indicates p<0.05, ** indicates p<0.005, and *** indicates p<0.0005 by unpaired two tailed students t-test comparing male and female samples or samples with or without heat shock.


A . PCR analysis of the human H49K variant of the ATPIF1 and rtTA constructs in wild - type ( wt ) , ACTA1 - rtTA ( T ) , ATPIF1H49K - TRE ( H ) or double transgenic ( T / H ) mice .

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A. PCR analysis of the human H49K variant of the ATPIF1 and rtTA constructs in wild-type (wt), ACTA1-rtTA (T), ATPIF1H49K-TRE (H) or double transgenic (T/H) mice.


( B WB expression of the human ( h ) ATPIF1 protein in Skm ( B extracts . hATPIF1 is only expressed in Skm from ATPIF1H49K | T / H mice . HSP60 are shown as loading controls . n = 3 mice / genotype .

(B WB expression of the human (h) ATPIF1 protein in Skm (B extracts. hATPIF1 is only expressed in Skm from ATPIF1H49K|T/H mice. HSP60 are shown as loading controls. n= 3 mice/genotype.


( C ) Immunofluorescent staining of wt and ATPIF1H49K | T / H hindlimb muscle slices with hATPIF1 and βF1 antibodies . Images are representative of 3 mice / genotype , 5 fields / mouse . hATPIF1 H49K is only expressed in T / H mice .

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(C) Immunofluorescent staining of wt and ATPIF1H49K|T/H hindlimb muscle slices with hATPIF1 and βF1 antibodies. Images are representative of 3 mice/genotype, 5 fields/mouse. hATPIF1 H49K is only expressed in T/H mice.


D ) WB expression of the human + mouse ( m + h ) ATPIF1 protein in Skm D ) , brain , liver , and WAT ( D ) extracts . hATPIF1 is only expressed in Skm from ATPIF1H49K | T / H mice . α - tubulin are shown as loading controls . n = 3 mice / genotype .

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D) WB expression of the human + mouse (m+h) ATPIF1 protein in Skm D), brain, liver, and WAT (D) extracts. hATPIF1 is only expressed in Skm from ATPIF1H49K|T/H mice. α-tubulin are shown as loading controls. n= 3 mice/genotype.


E . Polarographic profiles of isolated mitochondria from wt ( lower trace ) and ATPIF1H49K | T / H ( upper trace ) animals . Histograms show a reduction in state 3 respiration consistent with the inhibition of ATP synthase in ATPIF1H49K | T / H mice . Bars are the mean ± s . e . m . of n = 3 mice / genotype , 3 traces / mouse ; OL , oligomycin ; Ant A , antimycin A .

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E. Polarographic profiles of isolated mitochondria from wt (lower trace) and ATPIF1H49K|T/H (upper trace) animals. Histograms show a reduction in state 3 respiration consistent with the inhibition of ATP synthase in ATPIF1H49K|T/H mice. Bars are the mean ± s.e.m. of n= 3 mice/genotype, 3 traces/mouse; OL, oligomycin; Ant A, antimycin A.


( F , G ) Quantitative proteomic analysis ( iTRAQ ) of hindlimb Skm from wt and LowOXPHOS mice . The pie charts ( F ) and volcano plot ( G ) present the upregulated ( log2 [ LowOXPHOS / wt ] ≥ 1 , red ) or downregulated ( log2 [ LowOXPHOS / wt ] ≤ - 1 , blue ) proteins . A ( - ) log p value of > 4 was considered statistically significant . Data information : Data are the mean ± s . e . m . of n = 12 animals / genotype measured in 2 different iTRAQ analysis * p < 0 . 05 when compared to wt by ANOVA and Student ' s t - test .

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(F, G) Quantitative proteomic analysis (iTRAQ) of hindlimb Skm from wt and LowOXPHOS mice. The pie charts (F) and volcano plot (G) present the upregulated (log2[LowOXPHOS/wt]≥1, red) or downregulated (log2[LowOXPHOS/wt]≤-1, blue) proteins. A (-)log p value of >4 was considered statistically significant. Data information: Data are the mean ± s.e.m. of n= 12 animals/ genotype measured in 2 different iTRAQ analysis *p <0.05 when compared to wt by ANOVA and Student's t-test.


H . Cytoscape representation of the most upregulated proteins in Skm from LowOXPHOS mice . Data information : Data are the mean ± s . e . m . of n = 12 animals / genotype measured in 2 different iTRAQ analysis * p < 0 . 05 when compared to wt by ANOVA and Student ' s t - test .

H. Cytoscape representation of the most upregulated proteins in Skm from LowOXPHOS mice. Data information: Data are the mean ± s.e.m. of n= 12 animals/ genotype measured in 2 different iTRAQ analysis *p <0.05 when compared to wt by ANOVA and Student's t-test.


K . Skm iTRAQ ratio of proteins from BCAA catabolism . Higher intensities of red or blue colors represent higher or lower LowOXPHOS / wt expression ratios , respectively . Data information : Data are the mean ± s . e . m . of n = 12 animals / genotype measured in 2 different iTRAQ analysis * p < 0 . 05 when compared to wt by ANOVA and Student ' s t - test .

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K. Skm iTRAQ ratio of proteins from BCAA catabolism. Higher intensities of red or blue colors represent higher or lower LowOXPHOS/wt expression ratios, respectively. Data information: Data are the mean ± s.e.m. of n= 12 animals/ genotype measured in 2 different iTRAQ analysis *p <0.05 when compared to wt by ANOVA and Student's t-test.


A . Representative images of mice and body weight graph following the expression of mitochondrial ATPIF1H49K ( days of doxycycline ) ( wt , n = 12 ; LowOXPHOS , n = 12 ) .

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A. Representative images of mice and body weight graph following the expression of mitochondrial ATPIF1H49K (days of doxycycline) (wt, n= 12; LowOXPHOS, n= 12).


B . Representative images of hindlimb muscles . Arrows point to the soleus . ( C , D ) Transversal slices of red fibers from soleus stained with hematoxylin / eosin ( C ) . Higher im - WAT infiltrations in LowOXPHOS mice are shown . Quantifications in D ( wt , n = 6 ; LowOXPHOS , n = 6 ; 10 fields / mouse ) .

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B. Representative images of hindlimb muscles. Arrows point to the soleus. (C, D) Transversal slices of red fibers from soleus stained with hematoxylin/eosin (C). Higher im-WAT infiltrations in LowOXPHOS mice are shown. Quantifications in D (wt, n= 6; LowOXPHOS, n= 6; 10 fields/mouse).


iTRAQ ratio ( E ) of Skm proteins from de novo lipid synthesis . The expression of ATP citrate lyase ( ACLY ) , acetyl - CoA carboxylase ( ACAC ) , fatty acid synthase ( FASN ) are shown . Two samples per condition ; each sample contains protein extracts from 3 mice . In E , a higher intensity of red color represents a higher LowOXPHOS / wt expression ratio .

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iTRAQ ratio (E) of Skm proteins from de novo lipid synthesis. The expression of ATP citrate lyase (ACLY), acetyl-CoA carboxylase (ACAC), fatty acid synthase (FASN) are shown. Two samples per condition; each sample contains protein extracts from 3 mice. In E, a higher intensity of red color represents a higher LowOXPHOS/wt expression ratio.


representative WB expression ( F ) of Skm proteins from de novo lipid synthesis . The expression of ATP citrate lyase ( ACLY ) , acetyl - CoA carboxylase ( ACAC ) , fatty acid synthase ( FASN ) and their phosphorylation ( p ) are shown . Two samples per condition ; each sample contains protein extracts from 3 mice . GAPDH is shown as a loading control . Quantifications in lateral histograms ( wt , n = 6 ; LowOXPHOS , n = 6 ) .

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representative WB expression (F) of Skm proteins from de novo lipid synthesis. The expression of ATP citrate lyase (ACLY), acetyl-CoA carboxylase (ACAC), fatty acid synthase (FASN) and their phosphorylation (p) are shown. Two samples per condition; each sample contains protein extracts from 3 mice. GAPDH is shown as a loading control. Quantifications in lateral histograms (wt, n= 6; LowOXPHOS, n= 6).


G . Immunocapture ( IP ) of Skm acetylated proteins blotted with anti - ACLY antibody .

G. Immunocapture (IP) of Skm acetylated proteins blotted with anti-ACLY antibody.


H . Skm acetyl - CoA amounts ( wt , n = 8 ; LowOXPHOS , n = 8 ) . Data information : Bars are the mean ± s . e . m . of the indicated ( n ) mice / genotype * p < 0 . 05 when compared to wt by ANOVA and Student ' s t - test .

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H. Skm acetyl-CoA amounts (wt, n= 8; LowOXPHOS, n= 8). Data information: Bars are the mean ± s.e.m. of the indicated (n) mice/genotype *p <0.05 when compared to wt by ANOVA and Student's t-test.


J . Myocyte rates of aerobic glycolysis to lactate production . Bars are the mean ± s . e . m . of n = 3 experiments , 9 replicas / condition . Data information : Bars are the mean ± s . e . m . of the indicated ( n ) mice / genotype * p < 0 . 05 when compared to wt by ANOVA and Student ' s t - test .

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J. Myocyte rates of aerobic glycolysis to lactate production. Bars are the mean ± s.e.m. of n= 3 experiments, 9 replicas/condition. Data information: Bars are the mean ± s.e.m. of the indicated (n) mice/genotype *p <0.05 when compared to wt by ANOVA and Student's t-test.


( K , L ) . 14C ( u ) - glucose uptake ( K ) and oxidation to CO2 ( L ) in myocytes expressing or not the ATP synthase inhibitor ATPIF1H49K . Bars are the mean ± s . e . m . of n = 3 experiments , 6 replicas / condition . Data information : Bars are the mean ± s . e . m . of the indicated ( n ) mice / genotype * p < 0 . 05 when compared to wt by ANOVA and Student ' s t - test .

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(K, L). 14C(u)-glucose uptake (K) and oxidation to CO2 (L) in myocytes expressing or not the ATP synthase inhibitor ATPIF1H49K. Bars are the mean ± s.e.m. of n= 3 experiments, 6 replicas/condition. Data information: Bars are the mean ± s.e.m. of the indicated (n) mice/genotype *p <0.05 when compared to wt by ANOVA and Student's t-test.


M . Representative WB expression of Skm PDH and its phosphorylation . Two samples per condition ; each sample contains extracts from 3 mice . Quantification in histograms ( wt , n = 6 ; LowOXPHOS , n = 6 ) . Data information : Bars are the mean ± s . e . m . of the indicated ( n ) mice / genotype * p < 0 . 05 when compared to wt by ANOVA and Student ' s t - test .

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M. Representative WB expression of Skm PDH and its phosphorylation. Two samples per condition; each sample contains extracts from 3 mice. Quantification in histograms (wt, n= 6; LowOXPHOS, n= 6). Data information: Bars are the mean ± s.e.m. of the indicated (n) mice/genotype *p <0.05 when compared to wt by ANOVA and Student's t-test.


N . Skm levels of lactate ( wt , n = 6 ; LowOXPHOS , n = 6 ) . Data information : Bars are the mean ± s . e . m . of the indicated ( n ) mice / genotype * p < 0 . 05 when compared to wt by ANOVA and Student ' s t - test .

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N. Skm levels of lactate (wt, n= 6; LowOXPHOS, n= 6). Data information: Bars are the mean ± s.e.m. of the indicated (n) mice/genotype *p <0.05 when compared to wt by ANOVA and Student's t-test.


O . FFA β - oxidation in primary cultures of myocytes derived from wt or LowOXPHOS mouse hindlimbs . Bars are the mean ± s . e . m . of n = 3 experiments , 9 replicas / condition . Data information : Bars are the mean ± s . e . m . of the indicated ( n ) mice / genotype * p < 0 . 05 when compared to wt by ANOVA and Student ' s t - test .

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O. FFA β-oxidation in primary cultures of myocytes derived from wt or LowOXPHOS mouse hindlimbs. Bars are the mean ± s.e.m. of n= 3 experiments, 9 replicas/condition. Data information: Bars are the mean ± s.e.m. of the indicated (n) mice/genotype *p <0.05 when compared to wt by ANOVA and Student's t-test.


P . FFA amounts in Skm extracts ( wt , n = 8 ; LowOXPHOS , n = 8 ) . Data information : Bars are the mean ± s . e . m . of the indicated ( n ) mice / genotype * p < 0 . 05 when compared to wt by ANOVA and Student ' s t - test .

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P. FFA amounts in Skm extracts (wt, n= 8; LowOXPHOS, n= 8). Data information: Bars are the mean ± s.e.m. of the indicated (n) mice/genotype *p <0.05 when compared to wt by ANOVA and Student's t-test.


Transversal slices of soleus stained with hematoxylin / eosin ( Q ) . Arrows indicate LDs in LowOXPHOS mice . Images are representative of n = 4 mice / genotype , 10 images / mouse .

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Transversal slices of soleus stained with hematoxylin/eosin (Q). Arrows indicate LDs in LowOXPHOS mice. Images are representative of n= 4 mice/genotype, 10 images/mouse.


Oil Red O staining and enzymatic activity of respiratory CI in transverse contiguous slices ( R ) . Images are representative of n = 4 mice / genotype , 10 images / mouse . Note the colocalization of oxidative fibers with LDs ( R ) .

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Oil Red O staining and enzymatic activity of respiratory CI in transverse contiguous slices (R). Images are representative of n= 4 mice/genotype, 10 images/mouse. Note the colocalization of oxidative fibers with LDs (R).


S . Electron microscopy of transversal slices of the soleus . Intrafiber LDs surrounded by mitochondria in LowOXPHOS mice are shown . Histogram shows the quantifications of n = 4 animals / genotype , 10 images / animal .

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S. Electron microscopy of transversal slices of the soleus. Intrafiber LDs surrounded by mitochondria in LowOXPHOS mice are shown. Histogram shows the quantifications of n= 4 animals/genotype, 10 images/animal.


T . LD formation upon ATP synthase inhibition ( ATPIF1H49K ) in starved myocytes after 24 hours of palmitate supplementation in the presence or absence of BCAA . Blue : DAPI , nuclei ; green : BODIPY - positive LDs . Histograms show the quantification expressed as the number of LDs / nuclei . Bars are the mean ± s . e . m . of n = 3 experiments , 12 fields / condition . U . LD formation upon ATP synthase inhibition ( 5µM oligomycin ) in myocytes after 24 hours of palmitate supplementation . Histograms show the quantification expressed as fold of control of the BODIPY / DAPI fluorescence intensity . 7 fields / condition . Data information : Bars are the mean ± s . e . m . of the indicated ( n ) mice / genotype * p < 0 . 05 when compared to wt by ANOVA and Student ' s t - test .

O O 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 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

T. LD formation upon ATP synthase inhibition (ATPIF1H49K) in starved myocytes after 24 hours of palmitate supplementation in the presence or absence of BCAA. Blue: DAPI, nuclei; green: BODIPY-positive LDs. Histograms show the quantification expressed as the number of LDs/nuclei. Bars are the mean ± s.e.m. of n= 3 experiments, 12 fields/condition. U. LD formation upon ATP synthase inhibition (5µM oligomycin) in myocytes after 24 hours of palmitate supplementation. Histograms show the quantification expressed as fold of control of the BODIPY/DAPI fluorescence intensity. 7 fields/condition. Data information: Bars are the mean ± s.e.m. of the indicated (n) mice/genotype *p <0.05 when compared to wt by ANOVA and Student's t-test.


Plasma ( A ) BCAA levels in wt and LowOXPHOS mice ( wt , n = 4 ; LowOXPHOS , n = 4 ) . Bars are the mean ± s . e . m . of n = 3 experiments , 6 replicas / condition . Data information : Bars are the mean ± s . e . m . of the indicated ( n ) mice / genotype * p < 0 . 05 when compared to wt by ANOVA or Student ' s t - test .

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

Plasma (A) BCAA levels in wt and LowOXPHOS mice (wt, n= 4; LowOXPHOS, n= 4). Bars are the mean ± s.e.m. of n= 3 experiments, 6 replicas/condition. Data information: Bars are the mean ± s.e.m. of the indicated (n) mice/genotype *p <0.05 when compared to wt by ANOVA or Student's t-test.


Skm ( C ) BCAA levels in wt and LowOXPHOS mice ( wt , n = 4 ; LowOXPHOS , n = 4 ) . Bars are the mean ± s . e . m . of n = 3 experiments , 6 replicas / condition . Data information : Bars are the mean ± s . e . m . of the indicated ( n ) mice / genotype * p < 0 . 05 when compared to wt by ANOVA or Student ' s t - test .

Skm (C) BCAA levels in wt and LowOXPHOS mice (wt, n= 4; LowOXPHOS, n= 4). Bars are the mean ± s.e.m. of n= 3 experiments, 6 replicas/condition. Data information: Bars are the mean ± s.e.m. of the indicated (n) mice/genotype *p <0.05 when compared to wt by ANOVA or Student's t-test.


D , E ) . oxidation ( D ) and incorporation into lipids ( E ) in myocytes expressing or not ATPIF1H49K . Bars are the mean ± s . e . m . of n = 3 experiments , 6 replicas / condition . Data information : Bars are the mean ± s . e . m . of the indicated ( n ) mice / genotype * p < 0 . 05 when compared to wt by ANOVA or Student ' s t - test .

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

D, E). oxidation (D) and incorporation into lipids (E) in myocytes expressing or not ATPIF1H49K. Bars are the mean ± s.e.m. of n= 3 experiments, 6 replicas/condition. Data information: Bars are the mean ± s.e.m. of the indicated (n) mice/genotype *p <0.05 when compared to wt by ANOVA or Student's t-test.


F . Longitudinal ( left panels ) and transversal ( right panels ) slices of soleus stained with hematoxylin / eosin . im - WAT infiltrations and fiber shrinkage ( arrows ) in HFD - fed LowOXPHOS mice ( wt + HFD , n = 4 ; LowOXPHOS + HFD , n = 4 ; 10 fields / mouse ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Longitudinal (left panels) and transversal (right panels) slices of soleus stained with hematoxylin/eosin. im-WAT infiltrations and fiber shrinkage (arrows) in HFD-fed LowOXPHOS mice (wt + HFD, n= 4; LowOXPHOS + HFD, n= 4; 10 fields/mouse).


( G - I ) . Motor function tests . Rotarod ( G ; wt , n = 7 ; LowOXPHOS , n = 6 ) , Open Field ( H ; wt , n = 4 ; LowOXPHOS , n = 4 ) and gripforce ( after fatigue , I ; wt , n = 7 ; LowOXPHOS , n = 6 ) tests . In H , lines represent the movement of the mice in the cage . The histogram represents the relative time that wt and and LowOXPHOS mice expended in the corners . Data information : Bars are the mean ± s . e . m . of the indicated ( n ) mice / genotype * p < 0 . 05 when compared to wt by ANOVA or Student ' s t - test .

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

(G-I). Motor function tests. Rotarod (G; wt, n= 7; LowOXPHOS, n= 6), Open Field (H; wt, n= 4; LowOXPHOS, n= 4) and gripforce (after fatigue, I; wt, n= 7; LowOXPHOS, n= 6) tests. In H, lines represent the movement of the mice in the cage. The histogram represents the relative time that wt and and LowOXPHOS mice expended in the corners. Data information: Bars are the mean ± s.e.m. of the indicated (n) mice/genotype *p <0.05 when compared to wt by ANOVA or Student's t-test.


( J , K ) Quantitative Skm lipidomics at day 80 of HFD ( J ) . The color scale ( brown to blue ) in the heat - map represents LowOXPHOS / wt amounts of detailed lipid species . A p - value ≤ 0 . 05 was considered statistically significant ( red ) . In the lower heat - map ( K ) , the color scale ( yellow to blue ) highlights an increase in DAG in HFD - fed LowOXPHOS mice . Each sample is a pool from 4 mouse extracts . Histograms show the total amounts of Skm DAG ( wt , n = 8 ; LowOXPHOS , n = 8 ) . L . Blood triglycerides on the 80th day of HFD ( wt + HFD , n = 10 ; LowOXPHOS + HFD , n = 10 ) . Data information : Bars are the mean ± s . e . m . of the indicated ( n ) mice / genotype * p < 0 . 05 when compared to wt by ANOVA or Student ' s t - test .

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

(J, K) Quantitative Skm lipidomics at day 80 of HFD (J). The color scale (brown to blue) in the heat-map represents LowOXPHOS/wt amounts of detailed lipid species. A p-value ≤ 0.05 was considered statistically significant (red). In the lower heat-map (K), the color scale (yellow to blue) highlights an increase in DAG in HFD-fed LowOXPHOS mice. Each sample is a pool from 4 mouse extracts. Histograms show the total amounts of Skm DAG (wt, n= 8; LowOXPHOS, n= 8). L. Blood triglycerides on the 80th day of HFD (wt + HFD, n= 10; LowOXPHOS + HFD, n= 10). Data information: Bars are the mean ± s.e.m. of the indicated (n) mice/genotype *p <0.05 when compared to wt by ANOVA or Student's t-test.


M . Skm relative expression of the GLUT1 , GLUT3 , PPARβ / δ and PGC1α by qPCR ( wt , n = 5 ; LowOXPHOS , n = 5 ) . Data information : Bars are the mean ± s . e . m . of the indicated ( n ) mice / genotype * p < 0 . 05 when compared to wt by ANOVA or Student ' s t - test .

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

M. Skm relative expression of the GLUT1, GLUT3, PPARβ/δ and PGC1α by qPCR (wt, n= 5; LowOXPHOS, n= 5). Data information: Bars are the mean ± s.e.m. of the indicated (n) mice/genotype *p <0.05 when compared to wt by ANOVA or Student's t-test.


N . qPCR relative expression of myokines ( wt , n = 5 ; LowOXPHOS , n = 5 ) . Red or blue color represents higher or lower % expression , respectively , compared to that in wt . SPARC , osteonectin ; OSTN , musclin ; LIF , interleukin 6 family cytokine ; FKN , fractalkine ; FSTL1 , follistatin - like protein 1 ; FNDC5 , irisin ; ADIPOQ , adiponectin . Data information : Bars are the mean ± s . e . m . of the indicated ( n ) mice / genotype * p < 0 . 05 when compared to wt by ANOVA or Student ' s t - test .

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

N. qPCR relative expression of myokines (wt, n= 5; LowOXPHOS, n= 5). Red or blue color represents higher or lower % expression, respectively, compared to that in wt. SPARC, osteonectin; OSTN, musclin; LIF, interleukin 6 family cytokine; FKN, fractalkine; FSTL1, follistatin-like protein 1; FNDC5, irisin; ADIPOQ, adiponectin. Data information: Bars are the mean ± s.e.m. of the indicated (n) mice/genotype *p <0.05 when compared to wt by ANOVA or Student's t-test.


O . Blood glucose following the administration of HFD ( wt + HFD , n = 10 ; LowOXPHOS + HFD , n = 10 ) . T2D onset occurred on day 80 in wt and day 60 in LowOXPHOS mice .

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. Blood glucose following the administration of HFD (wt + HFD, n= 10; LowOXPHOS + HFD, n= 10). T2D onset occurred on day 80 in wt and day 60 in LowOXPHOS mice.


P . Insulin ( ITT ) and glucose ( GTT ) tolerance tests on day 60 of HFD ( wt + HFD , n = 10 ; LowOXPHOS + HFD , n = 10 ) . LowOXPHOS but not wt mice appeared diabetic .

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

P. Insulin (ITT) and glucose (GTT) tolerance tests on day 60 of HFD (wt + HFD, n= 10; LowOXPHOS + HFD, n= 10). LowOXPHOS but not wt mice appeared diabetic.


A . v - WAT iTRAQ ratio of proteins from BCAA catabolism ( wt , n = 4 ; LowOXPHOS , n = 4 ) . Higher intensities of blue colors represent lower LowOXPHOS / wt expression ratios .

A. v-WAT iTRAQ ratio of proteins from BCAA catabolism (wt, n= 4; LowOXPHOS, n= 4). Higher intensities of blue colors represent lower LowOXPHOS/wt expression ratios.


B . Odd chain FAs in Skm and WAT from wt ( n = 8 ) or LowOXPHOS ( n = 8 ) mice . Data information : Bars are the mean ± s . e . m . of the indicated ( n ) mice / genotype . * p < 0 . 05 when compared to wt by Student ' s t - test .

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

B. Odd chain FAs in Skm and WAT from wt (n= 8) or LowOXPHOS (n= 8) mice. Data information: Bars are the mean ± s.e.m. of the indicated (n) mice/genotype. *p <0.05 when compared to wt by Student's t-test.


Representative WB expression ( C ) of v - WAT proteins from lipid metabolism . Each sample contains extracts from 3 mice . The histogram shows the quantifications ( wt , n = 6 ; LowOXPHOS , n = 6 ) . Data information : Bars are the mean ± s . e . m . of the indicated ( n ) mice / genotype . * p < 0 . 05 when compared to wt by Student ' s t - test .

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

Representative WB expression (C) of v-WAT proteins from lipid metabolism. Each sample contains extracts from 3 mice. The histogram shows the quantifications (wt, n= 6; LowOXPHOS, n= 6). Data information: Bars are the mean ± s.e.m. of the indicated (n) mice/genotype. *p <0.05 when compared to wt by Student's t-test.


iTRAQ analysis ( D ) of v - WAT proteins from lipid metabolism . Each sample contains extracts from 3 mice . The histogram shows the quantifications ( wt , n = 6 ; LowOXPHOS , n = 6 ) .

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

iTRAQ analysis (D) of v-WAT proteins from lipid metabolism. Each sample contains extracts from 3 mice. The histogram shows the quantifications (wt, n= 6; LowOXPHOS, n= 6).


E . v - WAT weight expressed as a percentage of the wt ( wt , n = 12 ; LowOXPHOS , n = 12 ) . F . Quantitative lipidomics in v - WAT . The color scale ( brown to blue ) in the heat map represents LowOXPHOS / wt amounts of specific lipid species . A p - value ≤ 0 . 05 was considered statistically significant ( red ) . Each sample is a pool from 4 mouse extracts , n = 8 / genotype . Data information : Bars are the mean ± s . e . m . of the indicated ( n ) mice / genotype . * p < 0 . 05 when compared to wt by Student ' s t - test .

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

E. v-WAT weight expressed as a percentage of the wt (wt, n= 12; LowOXPHOS, n= 12). F. Quantitative lipidomics in v-WAT. The color scale (brown to blue) in the heat map represents LowOXPHOS/wt amounts of specific lipid species. A p-value ≤ 0.05 was considered statistically significant (red). Each sample is a pool from 4 mouse extracts, n= 8/genotype. Data information: Bars are the mean ± s.e.m. of the indicated (n) mice/genotype. *p <0.05 when compared to wt by Student's t-test.


G . v - WAT amounts of triacylglycerides ( TAGs ) and diacylglycerides ( DAGs ) ( wt , n = 8 ; LowOXPHOS , n = 8 ) . Data information : Bars are the mean ± s . e . m . of the indicated ( n ) mice / genotype . * p < 0 . 05 when compared to wt by Student ' s t - test .

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

G. v-WAT amounts of triacylglycerides (TAGs) and diacylglycerides (DAGs) (wt, n= 8; LowOXPHOS, n= 8). Data information: Bars are the mean ± s.e.m. of the indicated (n) mice/genotype. *p <0.05 when compared to wt by Student's t-test.


H . Higher saturated lipid species in LowOXPHOS mice compared to wt ( wt , n = 8 ; LowOXPHOS , n = 8 ) . Data information : Bars are the mean ± s . e . m . of the indicated ( n ) mice / genotype . * p < 0 . 05 when compared to wt by Student ' s t - test .

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

H. Higher saturated lipid species in LowOXPHOS mice compared to wt (wt, n= 8; LowOXPHOS, n= 8). Data information: Bars are the mean ± s.e.m. of the indicated (n) mice/genotype. *p <0.05 when compared to wt by Student's t-test.


I . MitoSox staining in myocytes expressing or not ATPIF1H49K . The right histogram shows the quantification of mitochondrial ROS . Bars are the mean ± s . e . m . of n = 3 experiments , 12 replicas / condition . Data information : Bars are the mean ± s . e . m . of the indicated ( n ) mice / genotype . * p < 0 . 05 when compared to wt by Student ' s t - test .

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

I. MitoSox staining in myocytes expressing or not ATPIF1H49K. The right histogram shows the quantification of mitochondrial ROS. Bars are the mean ± s.e.m. of n= 3 experiments, 12 replicas/condition. Data information: Bars are the mean ± s.e.m. of the indicated (n) mice/genotype. *p <0.05 when compared to wt by Student's t-test.


J . Representative WB of Skm lipid peroxidation and redox system proteins . Two samples per condition are shown . Each sample contains extracts from 3 mice . Histograms represent quantification ( wt , n = 6 ; LowOXPHOS , n = 6 ) . 4 - Hydroxynonenal ( 4HN ) , peroxiredoxin 2 , 3 and 6 ( PRX ) , superoxide dismutase 1 and 2 ( SOD ) , catalase ( CATA ) and glutathione reductase ( GSR ) immunoblots are shown . βF1 is presented as a loading control . Data information : Bars are the mean ± s . e . m . of the indicated ( n ) mice / genotype . * p < 0 . 05 when compared to wt by Student ' s t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative WB of Skm lipid peroxidation and redox system proteins. Two samples per condition are shown. Each sample contains extracts from 3 mice. Histograms represent quantification (wt, n= 6; LowOXPHOS, n= 6). 4-Hydroxynonenal (4HN), peroxiredoxin 2, 3 and 6 (PRX), superoxide dismutase 1 and 2 (SOD), catalase (CATA) and glutathione reductase (GSR) immunoblots are shown. βF1 is presented as a loading control. Data information: Bars are the mean ± s.e.m. of the indicated (n) mice/genotype. *p <0.05 when compared to wt by Student's t-test.


K . Representative WB of Skm OXPHOS and related system proteins . Two samples per condition are shown . Each sample contains extracts from 3 mice . Histograms represent quantification ( wt , n = 6 ; LowOXPHOS , n = 6 ) . NDUFA9 ( CI ) , SDHA and B ( CII ) , ETF subunits A and B , ETFDH , CoreII ( CIII ) , subunit 1 ( CIV ) , βF1 ( CV ) and hATPIF1 immunoblots are shown . Tubulin is presented as a loading control . Data information : Bars are the mean ± s . e . m . of the indicated ( n ) mice / genotype . * p < 0 . 05 when compared to wt by Student ' s t - test .

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

K. Representative WB of Skm OXPHOS and related system proteins. Two samples per condition are shown. Each sample contains extracts from 3 mice. Histograms represent quantification (wt, n= 6; LowOXPHOS, n= 6). NDUFA9 (CI), SDHA and B (CII), ETF subunits A and B, ETFDH, CoreII (CIII), subunit 1 (CIV), βF1(CV) and hATPIF1 immunoblots are shown. Tubulin is presented as a loading control. Data information: Bars are the mean ± s.e.m. of the indicated (n) mice/genotype. *p <0.05 when compared to wt by Student's t-test.


L . Skm iTRAQ ratio of FADH2 - binding proteins ( wt , n = 12 ; LowOXPHOS , n = 12 ) . A higher intensity of red color represents a higher LowOXPHOS / wt expression ratio .

O O O O O O O O O O O O O O O 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. Skm iTRAQ ratio of FADH2-binding proteins (wt, n= 12; LowOXPHOS, n= 12). A higher intensity of red color represents a higher LowOXPHOS/wt expression ratio.


M . Oxidized FAD levels and the reduced / oxidized FAD ratio in Skm extracts ( wt , n = 8 ; LowOXPHOS n = 8 ) . Data information : Bars are the mean ± s . e . m . of the indicated ( n ) mice / genotype . * p < 0 . 05 when compared to wt by Student ' s t - test .

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

M. Oxidized FAD levels and the reduced/oxidized FAD ratio in Skm extracts (wt, n= 8; LowOXPHOS n= 8). Data information: Bars are the mean ± s.e.m. of the indicated (n) mice/genotype. *p <0.05 when compared to wt by Student's t-test.


A . Enzymatic activity of respiratory complex CII in Skm isolated mitochondria from wt and LowOXPHOS mice fed with chow or HFD ( wt , n = 4 ; LowOXPHOS , n = 4 ; wt + HFD , n = 4 ; LowOXPHOS + HFD , n = 4 ) .

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

A. Enzymatic activity of respiratory complex CII in Skm isolated mitochondria from wt and LowOXPHOS mice fed with chow or HFD (wt, n= 4; LowOXPHOS, n= 4; wt + HFD, n= 4; LowOXPHOS + HFD, n= 4).


B . Polarographic profiles of isolated mitochondria from wt ( lower trace ) and LowOXPHOS ( upper trace ) animals using succinate ( left graph ) or palmitoyl - carnitine ( right graph ) as a substrate . Quantification of maximal respiration in the right histogram . Bars are the mean ± s . e . m . of n = 3 mice / genotype , 3 traces / mouse ; OL , oligomycin ; Ant A , antimycin A . Data information : * , # p < 0 . 05 when compared to wt or LowOXPHOS , respectively , by ANOVA and Student ' s t - test .

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

B. Polarographic profiles of isolated mitochondria from wt (lower trace) and LowOXPHOS (upper trace) animals using succinate (left graph) or palmitoyl-carnitine (right graph) as a substrate. Quantification of maximal respiration in the right histogram. Bars are the mean ± s.e.m. of n= 3 mice/genotype, 3 traces/mouse; OL, oligomycin; Ant A, antimycin A. Data information: *, # p <0.05 when compared to wt or LowOXPHOS, respectively, by ANOVA and Student's t-test.


( C - E ) 2D - PAGE of Skm extracts ( C , D ) and acetylated proteins ( E ) from wt ( n = 4 ) and LowOXPHOS ( n = 3 ) mice . The isoelectric point ( pI ) of the CII subunits SDHB ( C ) and SDHA ( D ) was calculated by protein migration in pH 3 - 10 NL strips .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E) 2D-PAGE of Skm extracts (C, D) and acetylated proteins (E) from wt (n= 4) and LowOXPHOS (n= 3) mice. The isoelectric point (pI) of the CII subunits SDHB (C) and SDHA (D) was calculated by protein migration in pH 3-10 NL strips.


F . Immunocapture ( IP ) of SDHA blotted with anti - acetyl - K antibody ( upper panel ) and of acetylated proteins blotted with anti - SDHA antibody ( middle panel ) in Skm isolated mitochondria from wt ( n = 3 ) and LowOXPHOS ( n = 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 O O O O

F. Immunocapture (IP) of SDHA blotted with anti-acetyl-K antibody (upper panel) and of acetylated proteins blotted with anti-SDHA antibody (middle panel) in Skm isolated mitochondria from wt (n= 3) and LowOXPHOS (n= 3) mice.


I . MitoSox staining in myocytes expressing or not ATPIF1H49K . The left scheme illustrates where each ETC inhibitor works . The right histogram shows the quantification of mitochondrial ROS . Bars are the mean ± s . e . m . of n = 3 experiments , 12 replicas / condition . Data information : * , # p < 0 . 05 when compared to wt or LowOXPHOS , respectively , by ANOVA and Student ' s t - test .

O O O O O 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

I. MitoSox staining in myocytes expressing or not ATPIF1H49K. The left scheme illustrates where each ETC inhibitor works. The right histogram shows the quantification of mitochondrial ROS. Bars are the mean ± s.e.m. of n= 3 experiments, 12 replicas/condition. Data information: *, # p <0.05 when compared to wt or LowOXPHOS, respectively, by ANOVA and Student's t-test.


J . MitoSox staining in myocytes expressing or not ATPIF1H49K . The left scheme illustrates where each CII inhibitor works . The right histogram shows the quantification of mitochondrial ROS . Bars are the mean ± s . e . m . of n = 3 experiments , 12 replicas / condition . Data information : * , # p < 0 . 05 when compared to wt or LowOXPHOS , respectively , by ANOVA and Student ' s t - test .

J. MitoSox staining in myocytes expressing or not ATPIF1H49K. The left scheme illustrates where each CII inhibitor works. The right histogram shows the quantification of mitochondrial ROS. Bars are the mean ± s.e.m. of n= 3 experiments, 12 replicas/condition. Data information: *, # p <0.05 when compared to wt or LowOXPHOS, respectively, by ANOVA and Student's t-test.


K . Representative blue native immunoblots ( BN ) and clear native in - gel activity ( CN ) of Skm mitochondrial membrane proteins from wt ( n = 3 ) and LowOXPHOS ( n = 3 ) 2 - month - old mice . The migration of the respiratory complexes / supercomplexes CI - V is indicated . Subassembly of CI ( with no activity , suggesting degradation ) was observed in LowOXPHOS mice . VDAC is shown as a loading control .

K. Representative blue native immunoblots (BN) and clear native in-gel activity (CN) of Skm mitochondrial membrane proteins from wt (n= 3) and LowOXPHOS (n= 3) 2-month-old mice. The migration of the respiratory complexes/supercomplexes CI-V is indicated. Subassembly of CI (with no activity, suggesting degradation) was observed in LowOXPHOS mice. VDAC is shown as a loading control.


( A , B ) The schematic illustrates the screening process of 702 drugs related to the mitochondrial respiratory capacity assessed by Seahorse XFe96 in C2C12 myocytes . Palmitate is used as a substrate . A total of 41 hits were identified . Maximum respiration ( Max R , % of untreated cells ) of compounds categorized by their therapeutic use ( 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 O O O O O O O O O O O O O O O O O O O O O

(A, B) The schematic illustrates the screening process of 702 drugs related to the mitochondrial respiratory capacity assessed by Seahorse XFe96 in C2C12 myocytes. Palmitate is used as a substrate. A total of 41 hits were identified. Maximum respiration (Max R, % of untreated cells) of compounds categorized by their therapeutic use (B).


( C , D ) The schematic illustrates the screening process of the 41 hits on FFA β - oxidation using 9 , 103H ( N ) palmitic acid as a substrate in C2C12 myocytes . Ten enhancers were identified . D . Correlation between the maximum respiration ( x ) and palmitate β - oxidation ( y ) . Colors identify classes of pharmaceuticals as in B . Edaravone was selected as a hit .

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

(C, D) The schematic illustrates the screening process of the 41 hits on FFA β-oxidation using 9,103H(N) palmitic acid as a substrate in C2C12 myocytes. Ten enhancers were identified. D. Correlation between the maximum respiration (x) and palmitate β-oxidation (y). Colors identify classes of pharmaceuticals as in B. Edaravone was selected as a hit.


E . Representative respiratory profile of C2C12 myocytes treated ( green trace ) or not treated ( black trace ) with 2 µM edaravone . The CPT1 inhibitor etomoxir was used as a negative control . OCR , oxygen consumption rate ; OL , oligomycin ; DNP , 2 , 4 - dinitrophenol ; Rot , rotenone ; Ant A , antimycin A . Quantification in right histogram . Bars are the mean ± s . e . m . of 12 replicas / condition . Data information ; 0 . 05 when compared to wt by ANOVA and Student ' s t - test .

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

E. Representative respiratory profile of C2C12 myocytes treated (green trace) or not treated (black trace) with 2 µM edaravone. The CPT1 inhibitor etomoxir was used as a negative control. OCR, oxygen consumption rate; OL, oligomycin; DNP, 2,4-dinitrophenol; Rot, rotenone; Ant A, antimycin A. Quantification in right histogram. Bars are the mean ± s.e.m. of 12 replicas/condition. Data information ;0.05 when compared to wt by ANOVA and Student's t-test.


( F , G ) FFA β - oxidation in C2C12 myocytes transfected with CRL or ATPIF1H49K plasmids ( F ) and in primary myotubes from wt and LowOXPHOS mice ( G ) , treated ( green bars ) or not with 2 µM edaravone . Bars are the mean ± s . e . m . of n = 3 experiments , 9 replicas / condition . Data information : * p < 0 . 05 when compared to wt by ANOVA and Student ' s t - test .

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

(F, G) FFA β-oxidation in C2C12 myocytes transfected with CRL or ATPIF1H49K plasmids (F) and in primary myotubes from wt and LowOXPHOS mice (G), treated (green bars) or not with 2 µM edaravone. Bars are the mean ± s.e.m. of n= 3 experiments, 9 replicas/condition. Data information: *p <0.05 when compared to wt by ANOVA and Student's t-test.


A . Mitochondrial ROS after 3 hours treatment with 2 μM edaravone ( green bars ) , 10 nM MitoQ ( purple bars ) or 1 mM NAC ( blue bars ) . Bars are the mean ± s . e . m . of n = 3 experiments , 9 replicas / condition .

O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O 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. Mitochondrial ROS after 3 hours treatment with 2 μM edaravone (green bars), 10 nM MitoQ (purple bars) or 1 mM NAC (blue bars). Bars are the mean ± s.e.m. of n= 3 experiments, 9 replicas/condition.


B . Representative WB expression of Skm ROS system proteins in mice administered ( 30 days ) edaravone . Two samples per condition are shown . Each sample contains extracts from 3 mice ( wt + edaravone , n = 6 ; LowOXPHOS + edaravone n = 6 ) . Peroxiredoxin 2 and 3 ( PRX ) , catalase ( CATA ) and glutathione reductase ( GSR ) immunoblots are shown . GAPDH is presented as a loading 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

B. Representative WB expression of Skm ROS system proteins in mice administered (30 days) edaravone. Two samples per condition are shown. Each sample contains extracts from 3 mice (wt + edaravone, n= 6; LowOXPHOS + edaravone n= 6). Peroxiredoxin 2 and 3 (PRX), catalase (CATA) and glutathione reductase (GSR) immunoblots are shown. GAPDH is presented as a loading control.


C . qPCR relative expression of TNFα and PGC1α in myocytes treated for 24 h with 2μM edaravone ( green bars ) , 10nM MitoQ ( purple bars ) or 1 mM NAC ( blue bars ) . 6 replicas / condition .

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

C. qPCR relative expression of TNFα and PGC1α in myocytes treated for 24 h with 2μM edaravone (green bars), 10nM MitoQ (purple bars) or 1 mM NAC (blue bars). 6 replicas/condition.


Representative WB expression of Skm de novo lipid synthesis ( D ) One month of edaravone treatment downregulated FASN and ACLY in both wt and LowOXPHOS mice ( D ) Two samples per condition are shown . Each sample contains extracts from 3 mice . βF1 is presented as a loading control . Quantifications in lateral histograms ( wt , n = 6 ; LowOXPHOS , n = 6 ; wt + edaravone , n = 6 ; LowOXPHOS + edaravone , n = 6 ) .

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

Representative WB expression of Skm de novo lipid synthesis (D) One month of edaravone treatment downregulated FASN and ACLY in both wt and LowOXPHOS mice (D) Two samples per condition are shown. Each sample contains extracts from 3 mice. βF1 is presented as a loading control. Quantifications in lateral histograms (wt, n= 6; LowOXPHOS, n= 6; wt + edaravone, n= 6; LowOXPHOS + edaravone, n= 6).


Representative OXPHOS ( E ) proteins . One month of edaravone treatment in both wt and LowOXPHOS mice and rewired the upregulation of ETF - A , ETF - B and SDHA proteins to the levels of wt ( E ) . Two samples per condition are shown . Each sample contains extracts from 3 mice . Tubulin is presented as a loading control . Quantifications in lateral histograms ( wt , n = 6 ; LowOXPHOS , n = 6 ; wt + edaravone , n = 6 ; LowOXPHOS + edaravone , n = 6 ) .

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

Representative OXPHOS (E) proteins. One month of edaravone treatment in both wt and LowOXPHOS mice and rewired the upregulation of ETF-A, ETF-B and SDHA proteins to the levels of wt (E). Two samples per condition are shown. Each sample contains extracts from 3 mice. Tubulin is presented as a loading control. Quantifications in lateral histograms (wt, n= 6; LowOXPHOS, n= 6; wt + edaravone, n= 6; LowOXPHOS + edaravone, n= 6).


F . 2D - PAGE of Skm extracts from wt ( n = 3 ) and LoxOXPHOS ( n = 3 ) mice treated with edaravone . The pI of SDHA , calculated by protein migration in pH 3 - 10 NL strips , was the same for both genotypes .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 2D-PAGE of Skm extracts from wt (n= 3) and LoxOXPHOS (n= 3) mice treated with edaravone. The pI of SDHA, calculated by protein migration in pH 3-10 NL strips, was the same for both genotypes.


G . Representative blue native immunoblots ( BN ) of Skm mitochondrial membrane proteins from 2 - month - old mice treated with edaravone for 30 days ( wt + edaravone , n = 3 ; LowOXPHOS + edaravone , n = 3 ) . The migration of the respiratory complexes / supercomplexes CI - CIV and hATPIF1 is indicated . VDAC is shown as a loading control .

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

G. Representative blue native immunoblots (BN) of Skm mitochondrial membrane proteins from 2-month-old mice treated with edaravone for 30 days (wt + edaravone, n= 3; LowOXPHOS + edaravone, n= 3). The migration of the respiratory complexes/supercomplexes CI-CIV and hATPIF1 is indicated. VDAC is shown as a loading control.


H . Mouse body weight following the administration of HFD and edaravone . Data information : H - L : wt + HFD , n = 5 ; LowOXPHOS + HFD n = 4 ; wt + HFD + edaravone , n = 10 ; LowOXPHOS + HFD + edaravone n = 9 . Bars are the mean ± s . e . m . of the indicated ( n ) mice / genotype . * p < 0 . 05 when compared to wt by ANOVA and Student ' s t - test .

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

H. Mouse body weight following the administration of HFD and edaravone. Data information: H-L: wt + HFD, n= 5; LowOXPHOS + HFD n= 4; wt + HFD + edaravone, n= 10; LowOXPHOS + HFD + edaravone n= 9. Bars are the mean ± s.e.m. of the indicated (n) mice/genotype. *p <0.05 when compared to wt by ANOVA and Student's t-test.


I . v - WAT amounts at day 60th of HFD in mice treated with edaravone . Data information : H - L : wt + HFD , n = 5 ; LowOXPHOS + HFD n = 4 ; wt + HFD + edaravone , n = 10 ; LowOXPHOS + HFD + edaravone n = 9 . Bars are the mean ± s . e . m . of the indicated ( n ) mice / genotype . * p < 0 . 05 when compared to wt by ANOVA and Student ' s t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. v-WAT amounts at day 60th of HFD in mice treated with edaravone. Data information: H-L: wt + HFD, n= 5; LowOXPHOS + HFD n= 4; wt + HFD + edaravone, n= 10; LowOXPHOS + HFD + edaravone n= 9. Bars are the mean ± s.e.m. of the indicated (n) mice/genotype. *p <0.05 when compared to wt by ANOVA and Student's t-test.


J . Insulin ( ITT ) and glucose ( GTT ) tolerance tests after 60 days of HFD in mice treated with edaravone . Data information : H - L : wt + HFD , n = 5 ; LowOXPHOS + HFD n = 4 ; wt + HFD + edaravone , n = 10 ; LowOXPHOS + HFD + edaravone n = 9 . Bars are the mean ± s . e . m . of the indicated ( n ) mice / genotype . * p < 0 . 05 when compared to wt by ANOVA and Student ' s t - test .

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

J. Insulin (ITT) and glucose (GTT) tolerance tests after 60 days of HFD in mice treated with edaravone. Data information: H-L: wt + HFD, n= 5; LowOXPHOS + HFD n= 4; wt + HFD + edaravone, n= 10; LowOXPHOS + HFD + edaravone n= 9. Bars are the mean ± s.e.m. of the indicated (n) mice/genotype. *p <0.05 when compared to wt by ANOVA and Student's t-test.


K . GTT after 80 days of HFD ( left ) and ITT after 90 days of HFD ( right ) in wt and LowOXPHOS mice treated with edaravone , showing that the compound has an improving effect itself . Data information : H - L : wt + HFD , n = 5 ; LowOXPHOS + HFD n = 4 ; wt + HFD + edaravone , n = 10 ; LowOXPHOS + HFD + edaravone n = 9 . Bars are the mean ± s . e . m . of the indicated ( n ) mice / genotype . * p < 0 . 05 when compared to wt by ANOVA and Student ' s t - test .

O O O O 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 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

K. GTT after 80 days of HFD (left) and ITT after 90 days of HFD (right) in wt and LowOXPHOS mice treated with edaravone, showing that the compound has an improving effect itself. Data information: H-L: wt + HFD, n= 5; LowOXPHOS + HFD n= 4; wt + HFD + edaravone, n= 10; LowOXPHOS + HFD + edaravone n= 9. Bars are the mean ± s.e.m. of the indicated (n) mice/genotype. *p <0.05 when compared to wt by ANOVA and Student's t-test.


L . Blood glucose following the administration of HFD . T2D onset occurred on day 90 in wt and LowOXPHOS mice treated with edaravone . Data information : H - L : wt + HFD , n = 5 ; LowOXPHOS + HFD n = 4 ; wt + HFD + edaravone , n = 10 ; LowOXPHOS + HFD + edaravone n = 9 . Bars are the mean ± s . e . m . of the indicated ( n ) mice / genotype . * p < 0 . 05 when compared to wt by ANOVA and Student ' s t - test .

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

L. Blood glucose following the administration of HFD. T2D onset occurred on day 90 in wt and LowOXPHOS mice treated with edaravone. Data information: H-L: wt + HFD, n= 5; LowOXPHOS + HFD n= 4; wt + HFD + edaravone, n= 10; LowOXPHOS + HFD + edaravone n= 9. Bars are the mean ± s.e.m. of the indicated (n) mice/genotype. *p <0.05 when compared to wt by ANOVA and Student's t-test.


( B ) Maximum intensity projections ( top ) and representative sections ( bottom ) of typical nuclei of the indicated cell types stained with DAPI . Scale bars , 3 μm .

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

(B) Maximum intensity projections (top) and representative sections (bottom) of typical nuclei of the indicated cell types stained with DAPI. Scale bars, 3 μm.


( C ) Areas of DAPI - dense regions ( top ) , distance of DAPI - dense regions from the nuclear periphery ( middle ) , and variance of DAPI signals ( bottom ) . The point marks the median while the thick and thin lines correspond to 66 % and 95 % intervals , respectively . Number of DAPI dense regions = 950 / 1450 / 839 / 1535 / 736 and number of slices = 90 / 115 / 95 / 135 / 110 for mESC / EpiLC / d2 / d4c7 mPGCLC / GSC . Significances are computed using Wilcoxon rank - sum tests , p - values from top to bottom 4 . 37e - 3 , 1 . 62e - 3 , 2 . 99e - 2 , 2 . 03e - 10 , 4 . 03e - 1 , < 2 . 2e - 16 , 1 . 31e - 3 , 8 . 94e - 3 , 1 . 06e - 4 , 5 . 62e - 2 , 4 . 63e - 5 , 7 . 65e - 13 . P - value symbol brackets : ∗ ∗ ∗ ∗ = [ 0 , 0 . 0001 ) ; ∗ ∗ ∗ = [ 0 . 0001 , 0 . 001 ] ; ∗ ∗ = [ 0 . 001 , 0 . 01 ) ; ∗ = [ 0 . 01 , 0 . 05 ) ; ns = [ 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Areas of DAPI-dense regions (top), distance of DAPI-dense regions from the nuclear periphery (middle), and variance of DAPI signals (bottom). The point marks the median while the thick and thin lines correspond to 66% and 95% intervals, respectively. Number of DAPI dense regions = 950/1450/839/1535/736 and number of slices = 90/115/95/135/110 for mESC/EpiLC/d2/d4c7 mPGCLC/GSC. Significances are computed using Wilcoxon rank-sum tests, p-values from top to bottom 4.37e-3, 1.62e-3, 2.99e-2, 2.03e-10, 4.03e-1, <2.2e-16, 1.31e-3, 8.94e-3, 1.06e-4, 5.62e-2, 4.63e-5, 7.65e-13. P-value symbol brackets: ∗∗∗∗ = [0, 0.0001); ∗∗∗ = [0.0001, 0.001]; ∗∗ = [0.001, 0.01); ∗ = [0.01, 0.05); ns = [0.05,


( D ) ( left ) Fluorescence in situ hybridization ( FISH ) against chromosome 16 ( red ) with DAPI staining ( grey ) . Z - stacked representative images are paired with magnified views . ( right ) Distributions of surface volumes for chr16 . The point marks the median while the thick and thin lines correspond to 66 % and 95 % intervals , respectively . Number of cells = 51 / 68 / 53 for mESC / EpiLC / GSC . Scale bars , 5 μm . Significances are computed using Wilcoxon rank - sum tests , p - values from left to right : 4 . 16e - 2 , 4 . 33e - 6 , 8 . 68e - 9 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) (left) Fluorescence in situ hybridization (FISH) against chromosome 16 (red) with DAPI staining (grey). Z-stacked representative images are paired with magnified views. (right) Distributions of surface volumes for chr16. The point marks the median while the thick and thin lines correspond to 66% and 95% intervals, respectively. Number of cells = 51/68/53 for mESC/EpiLC/GSC. Scale bars, 5 μm. Significances are computed using Wilcoxon rank-sum tests, p-values from left to right: 4.16e-2, 4.33e-6, 8.68e-9.


( H ) Western blot for lamin B1 in different cell types ( bottom ) and quantification normalized by β - actin ( top ) .

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(H) Western blot for lamin B1 in different cell types (bottom) and quantification normalized by β-actin (top).


( L ) ( top ) Representative images of FISH against major satellite repeats in EpiLCs and GSCs . Scale bars , 10 μm ; ( bottom ) percentage of the pericentromeres detached from the nuclear lamina in EpiLCs and GSCs . The point marks the median while the thick and thin lines correspond to 66 % and 95 % intervals , respectively . Number of cells = 18 / 22 for EpiLC / GSC .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) (top) Representative images of FISH against major satellite repeats in EpiLCs and GSCs. Scale bars, 10 μm; (bottom) percentage of the pericentromeres detached from the nuclear lamina in EpiLCs and GSCs. The point marks the median while the thick and thin lines correspond to 66% and 95% intervals, respectively. Number of cells = 18/22 for EpiLC/GSC.


( I ) ( left ) Western blot for G9a , GLP , Setdb1 and α - tubulin ; ( right ) quantification normalized by α - Tubulin .

O O O O O 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) (left) Western blot for G9a, GLP, Setdb1 and α-tubulin; (right) quantification normalized by α-Tubulin.


( K ) ( top ) FISH against the Y chromosome ; ( bottom ) sphericity of the Y chromosome FISH signals ; ( right ) distributions Y chromosome surface volumes . The point marks the median while the thick and thin lines correspond to 66 % and 95 % intervals , respectively . Number of cells = 89 / 76 / 69 for mESC / EpiLC / GSC . Scale bar , 10 μm . Significances are computed using Wilcoxon rank - sum tests , p - values

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

(K) (top) FISH against the Y chromosome; (bottom) sphericity of the Y chromosome FISH signals; (right) distributions Y chromosome surface volumes. The point marks the median while the thick and thin lines correspond to 66% and 95% intervals, respectively. Number of cells = 89/76/69 for mESC/EpiLC/GSC. Scale bar, 10 μm. Significances are computed using Wilcoxon rank-sum tests, p-values from


( B ) Maximum intensity projections ( top ) and representative sections ( bottom ) of typical nuclei of GSCs and GSCLCs stained with DAPI . Scale bars , 3 μm . ( C ) Areas of DAPI - dense regions ( left ) , distance of DAPI - dense regions from the nuclear periphery ( middle ) , and variance of DAPI signals ( right ) . The point marks the median while the thick and thin lines correspond to 66 % and 95 % intervals , respectively . Number of DAPI dense regions = 1535 / 736 / 1227 and number of slices = 135 / 110 / 120 for d4c7 mPGCLC / GSC / GSCLC . Significances are computed using Wilcoxon rank - sum tests , p - values from left to right : 2 . 03e - 10 , 1 . 69e - 9 , 0 . 123 , 0 . 00894 , 8 . 02e - 8 , 0 . 0707 , 7 . 65e - 13 , 0 . 417 , 2 . 07e - 12 .

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(B) Maximum intensity projections (top) and representative sections (bottom) of typical nuclei of GSCs and GSCLCs stained with DAPI. Scale bars, 3 μm. (C) Areas of DAPI-dense regions (left), distance of DAPI-dense regions from the nuclear periphery (middle), and variance of DAPI signals (right). The point marks the median while the thick and thin lines correspond to 66% and 95% intervals, respectively. Number of DAPI dense regions = 1535/736/1227 and number of slices = 135/110/120 for d4c7 mPGCLC/GSC/GSCLC. Significances are computed using Wilcoxon rank-sum tests, p-values from left to right: 2.03e-10, 1.69e-9, 0.123, 0.00894, 8.02e-8, 0.0707, 7.65e-13, 0.417, 2.07e-12.


F Representative BLI and histology of organotypic cultures with H2030 - BrM cancer cells treated with DMSO or decreasing concentrations of DEBIO - 0932 . Scale bar : 100 µm ; high magnification : 50 µm . G Quantification of the bioluminescence signal emitted by each condition shown in ( F ) at day 3 normalized by the initial value obtained at day 0 and normalized to the organotypic cultures treated with DMSO . Day 0 is considered 12 - 16h after the addition of cancer cells and treatment or DMSO . Values are shown in box - and - whisker plots where each dot is an organotypic culture and the line in the box corresponds to the median . The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value ( n = 8 DMSO , n = 8 BKM120 and n = 7 per concentration of DEBIO - 0932 - treated organotypic cultures , 2 independent experiments ) . P value was calculated using two - tailed t - test .

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

F Representative BLI and histology of organotypic cultures with H2030-BrM cancer cells treated with DMSO or decreasing concentrations of DEBIO-0932. Scale bar: 100 µm; high magnification: 50 µm. G Quantification of the bioluminescence signal emitted by each condition shown in (F) at day 3 normalized by the initial value obtained at day 0 and normalized to the organotypic cultures treated with DMSO. Day 0 is considered 12-16h after the addition of cancer cells and treatment or DMSO. Values are shown in box-and-whisker plots where each dot is an organotypic culture and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value (n=8 DMSO, n=8 BKM120 and n=7 per concentration of DEBIO-0932-treated organotypic cultures, 2 independent experiments). P value was calculated using two-tailed t-test.


B Representative images of organotypic cultures with established metastases with various glial components of the microenvironment labelled . Scale bar : 75 µm . Each individual condition was evaluated in several organotypic cultures ( 3 - 6 slices ) .

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B Representative images of organotypic cultures with established metastases with various glial components of the microenvironment labelled. Scale bar: 75 µm. Each individual condition was evaluated in several organotypic cultures (3-6 slices).


D Quantification of LDH levels in the conditioned media of organotypic slices cultured during 3 days relative to a lysate of the same preparation . Values are shown as mean + s . e . m . ( n = 3 organotypic cultures per experimental condition , 1 independent experiment ) .

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D Quantification of LDH levels in the conditioned media of organotypic slices cultured during 3 days relative to a lysate of the same preparation. Values are shown as mean + s.e.m. (n=3 organotypic cultures per experimental condition, 1 independent experiment).


A - C Immunofluorescence against HSP90 in mouse brains with established metastases . ( A ) HSP90 positive structures in areas not affected by the metastasis includes the medial habenula , where neurons co - localize with the chaperone . Scale bars : 100 µm ( low magnification ) , 50 µm ( medial habenula nucleus ) , 12 µm ( high magnification neurons ) . ( B ) Established metastases from different primary origins and oncogenomic profiles stained with HSP90 . Dotted lines delineate the metastasis ( cc : cancer cells ) . Scale bars : 75 µm . ( C ) Iba1 colocalizes with HSP90 within areas affected by metastases . BB : bisbenzamide . Scale bar : 75 µm ( low magnification ) , 12 µm ( high magnification ) .

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A-C Immunofluorescence against HSP90 in mouse brains with established metastases. (A) HSP90 positive structures in areas not affected by the metastasis includes the medial habenula, where neurons co-localize with the chaperone. Scale bars: 100 µm (low magnification), 50 µm (medial habenula nucleus), 12 µm (high magnification neurons). (B) Established metastases from different primary origins and oncogenomic profiles stained with HSP90. Dotted lines delineate the metastasis (cc: cancer cells). Scale bars: 75 µm. (C) Iba1 colocalizes with HSP90 within areas affected by metastases. BB: bisbenzamide. Scale bar: 75 µm (low magnification), 12 µm (high magnification).


E Representative human brain metastases showing different intensities or scores for HSP90 . Scale bar : 50 µm . F Quantification of HSP90 in human brain metastases . 59 out of 60 ( 98 % ) showed positive staining of HSP90 in the tumor , 15 ( 25 % ) scored with 3 ( strong ) , 36 ( 60 % ) with 2 ( moderate ) and 8 ( 13 % ) with 1 ( weak ) according to the signal intensity of HSP90 in the cytoplasm of cancer cells .

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E Representative human brain metastases showing different intensities or scores for HSP90. Scale bar: 50 µm. F Quantification of HSP90 in human brain metastases. 59 out of 60 (98%) showed positive staining of HSP90 in the tumor, 15 (25%) scored with 3 (strong), 36 (60%) with 2 (moderate) and 8 (13%) with 1 (weak) according to the signal intensity of HSP90 in the cytoplasm of cancer cells.


Representative human brain metastases showing different percentages of nuclear HSP90 . Scale bars : ( J ) 50 µm Black arrows point to cancer cells positive for HSP90 in the nucleus .

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Representative human brain metastases showing different percentages of nuclear HSP90. Scale bars: (J) 50 µm Black arrows point to cancer cells positive for HSP90 in the nucleus.


Representative human brain metastases showing different percentages of nuclear HSP90 . Scale bars : ( K ) low magnification : 100 µm ; high magnification : 10 µm .

Representative human brain metastases showing different percentages of nuclear HSP90. Scale bars: (K) low magnification: 100 µm; high magnification: 10 µm.


L Quantification of nuclear HSP90 in human brain metastases . 54 out of 60 samples ( 90 % ) showed positive nuclear HSP90 in the tumor . 27 ( 45 % ) showed 1 - 5 % ( moderate ) and 27 ( 45 % ) showed > 5 % ( high ) of nuclear HSP90 . M Quantification of nuclear HSP90 in human primary tumors . 19 out of 30 ( 63 % ) showed positive nuclear HSP90 in the tumor . 9 ( 30 % ) showed 1 - 5 % ( moderate ) and 10 ( 33 % ) showed > 5 % ( high ) of nuclear HSP90 .

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L Quantification of nuclear HSP90 in human brain metastases. 54 out of 60 samples (90%) showed positive nuclear HSP90 in the tumor. 27 (45%) showed 1-5% (moderate) and 27 (45%) showed >5% (high) of nuclear HSP90. M Quantification of nuclear HSP90 in human primary tumors. 19 out of 30 (63%) showed positive nuclear HSP90 in the tumor. 9 (30%) showed 1-5% (moderate) and 10 (33%) showed >5% (high) of nuclear HSP90.


B Representative in vivo and ex vivo images of vehicle and DEBIO - 0932 treated mice 5 weeks ( experimental endpoint ) after intracardiac inoculation of H2030 - BrM cells .

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B Representative in vivo and ex vivo images of vehicle and DEBIO-0932 treated mice 5 weeks (experimental endpoint) after intracardiac inoculation of H2030-BrM cells.


E Representative sections of brains from vehicle and DEBIO - 0932 treated mice in ( B - D ) . The dotted lines surround the metastases ( GFP + ) . Representative field of view of metastasis stained with GFP and cleaved caspase 3 . Scale bars : slices , 1mm ; cleaved caspase 3 , 50 µm . F Quantification of established metastases found in vehicle and DEBIO - 0932 treated brains from panel ( E ) . Values are shown in box - and - whisker plots where every dot represents a different brain and the line in the box corresponds to the median . The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value ( vehicle : n = 10 brains ; DEBIO - 0932 : n = 14 brains ) . P value was calculated using two - tailed t - test . G Quantification of number of cleaved caspase 3 ( CC3 + ) in cancer cells found in vehicle and DEBIO - 0932 treated brains from panel ( E ) . Values are shown in box - and - whisker plots where every dot is a metastatic lesion and the line in the box corresponds to the median . The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value ( n = 8 metastatic lesions from 4 brains per condition ) . P value was calculated using two - tailed t - test .

O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Representative sections of brains from vehicle and DEBIO-0932 treated mice in (B-D). The dotted lines surround the metastases (GFP+). Representative field of view of metastasis stained with GFP and cleaved caspase 3. Scale bars: slices,1mm; cleaved caspase 3, 50 µm. F Quantification of established metastases found in vehicle and DEBIO-0932 treated brains from panel (E). Values are shown in box-and-whisker plots where every dot represents a different brain and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value (vehicle: n=10 brains; DEBIO-0932: n=14 brains). P value was calculated using two-tailed t-test. G Quantification of number of cleaved caspase 3 (CC3+) in cancer cells found in vehicle and DEBIO-0932 treated brains from panel (E). Values are shown in box-and-whisker plots where every dot is a metastatic lesion and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value (n=8 metastatic lesions from 4 brains per condition). P value was calculated using two-tailed t-test.


I Representative BrM - PDOC stained with proliferation markers ( BrdU ) and markers of the microenvironment ( GFAP for astrocytes , Iba1 for microglia / macrophages ) . Scale bar : 50 µm .

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I Representative BrM-PDOC stained with proliferation markers (BrdU) and markers of the microenvironment (GFAP for astrocytes, Iba1 for microglia/ macrophages). Scale bar: 50 µm.


F qRT - PCR of H2030 - BrM brain metastases obtained from animals during neurosurgery compared to relapsed metastases from the corresponding animals . A panel of five genes related to HSP90 pathway is evaluated . Values are shown in box - and - whisker plots where every dot represents a different animal and the line in the box corresponds to the median . The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value ( n = 5 mice per experimental condition ) . P value was calculated using two - tailed t - test .

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F qRT-PCR of H2030-BrM brain metastases obtained from animals during neurosurgery compared to relapsed metastases from the corresponding animals. A panel of five genes related to HSP90 pathway is evaluated. Values are shown in box-and-whisker plots where every dot represents a different animal and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value (n=5 mice per experimental condition). P value was calculated using two-tailed t-test.


H Representative images of vehicle and DEBIO - 0932 treated mice after neurosurgery until experimental endpoint at 6 and 8 weeks for vehicle and DEBIO - 0932 treated mice , respectively .

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H Representative images of vehicle and DEBIO-0932 treated mice after neurosurgery until experimental endpoint at 6 and 8 weeks for vehicle and DEBIO-0932 treated mice, respectively.


C Volcano plot with deregulated proteins ( red : upregulated ; green : downregulated ) found in brain metastases treated with DEBIO - 0932 compared to DMSO ( n = 3 biological replicates ( mice ) per condition , n ≥ 12 brain metastases per mouse were pooled together ) . Proteins with a P value < 0 . 05 and a log2 ratio > 1 or < - 1 were defined as deregulated . Gray dotted lines indicate P value and log2 ratio cut offs . The names of the top deregulated proteins are shown .

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C Volcano plot with deregulated proteins (red: upregulated; green: downregulated) found in brain metastases treated with DEBIO-0932 compared to DMSO (n=3 biological replicates (mice) per condition, n≥12 brain metastases per mouse were pooled together). Proteins with a P value <0.05 and a log2 ratio >1 or <-1 were defined as deregulated. Gray dotted lines indicate P value and log2 ratio cut offs. The names of the top deregulated proteins are shown.


G Kaplan - Meier curves showing significant correlation between worse survival post - brain metastasis and high expression levels of the HSP90 signature ( AHR , DDA1 , UBE4B , GPATCH8 ) in a cohort of 45 breast cancer brain metastasis patients .

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G Kaplan-Meier curves showing significant correlation between worse survival post-brain metastasis and high expression levels of the HSP90 signature (AHR, DDA1, UBE4B, GPATCH8) in a cohort of 45 breast cancer brain metastasis patients.


H Representative images ( selected cases obtained from Fig EV6M ) and histological score of AHR , DDA1 and UBE4B in human brain metastases ( n = 16 ) according to the signal intensity of the corresponding protein in cancer cells .

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H Representative images (selected cases obtained from Fig EV6M) and histological score of AHR, DDA1 and UBE4B in human brain metastases (n=16) according to the signal intensity of the corresponding protein in cancer cells.


K Quantification of ex vivo BLI of brains and thoracic regions at the endpoint of the experiment . Values are shown in box - and - whisker plots where every dot represents a different animal and the line in the box corresponds to the median . The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value ( n = 9 shControl mice and n = 10 shAHR # 1 mice ) . P value was calculated using two - tailed t - test .

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K Quantification of ex vivo BLI of brains and thoracic regions at the endpoint of the experiment. Values are shown in box-and-whisker plots where every dot represents a different animal and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value (n=9 shControl mice and n=10 shAHR#1 mice). P value was calculated using two-tailed t-test.


N Representative images of primary tumors with high ( red dot ) or low ( grey dot ) UBE4B levels . A few cases of matched primary - metastases allowed to evaluate the HSP90 - dependent protein . Scale bar : : 100 µm .

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N Representative images of primary tumors with high (red dot) or low (grey dot) UBE4B levels. A few cases of matched primary-metastases allowed to evaluate the HSP90-dependent protein. Scale bar: : 100 µm.


P Kaplan - Meier curve comparing relapse - free survival of primary tumors with high and low values of UBE4B . P value was calculated using log - rank ( Mantel - Cox ) test .

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P Kaplan-Meier curve comparing relapse-free survival of primary tumors with high and low values of UBE4B. P value was calculated using log-rank (Mantel-Cox) test.


D Representative organotypic cultures from the experiment in panel ( C ) . RFP is an internal control probe labelling cancer cells independent of autophagy flux and GFP indicate GFP - LC3 + puncta . The dotted line in the upper panel delimits a high magnification area shown in the lower panel respect to the GFP signal derived from GFP - LC3 accumulation . Dotted lines in lower panel surround individual cancer cells . Asterisk labels the area in the cell magnified in the high magnification panel showing the GFP - LC3 + puncta . Scale bar : low magnificantion , 25 µm ; high magnification ( cells ) , 10 µm ; high magnification ( puncta ) , 2 . 5 µm . E Quantification of GFP - LC3 + vesicles per cell of the experiment in panel ( C ) . Values are shown in box - and - whisker plots where every dot represents a field of view of an organotypic culture and the line in the box corresponds to the median . The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value ( DMSO : n = 15 fields of view , 2232 cancer cells from 3 organotypic cultures ; DEBIO - 0932 : n = 20 fields of view , 3260 cancer cells from 4 organotypic cultures ) . P value was calculated using two - tailed t - test .

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D Representative organotypic cultures from the experiment in panel (C). RFP is an internal control probe labelling cancer cells independent of autophagy flux and GFP indicate GFP-LC3+ puncta. The dotted line in the upper panel delimits a high magnification area shown in the lower panel respect to the GFP signal derived from GFP-LC3 accumulation. Dotted lines in lower panel surround individual cancer cells. Asterisk labels the area in the cell magnified in the high magnification panel showing the GFP-LC3+ puncta. Scale bar: low magnificantion, 25 µm; high magnification (cells), 10 µm; high magnification (puncta), 2.5 µm. E Quantification of GFP-LC3+ vesicles per cell of the experiment in panel (C). Values are shown in box-and-whisker plots where every dot represents a field of view of an organotypic culture and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value (DMSO: n=15 fields of view, 2232 cancer cells from 3 organotypic cultures; DEBIO-0932: n=20 fields of view, 3260 cancer cells from 4 organotypic cultures). P value was calculated using two-tailed t-test.


A Representative Gli1ZF / DNA structure extrapolated from MD trajectories . Gli1ZF is shown as blue cartoon , residues involved in binding to DNA ( based on single point mutation study ) are shown as magenta sticks , and Zn ions as gray spheres .

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A Representative Gli1ZF/DNA structure extrapolated from MD trajectories. Gli1ZF is shown as blue cartoon, residues involved in binding to DNA (based on single point mutation study) are shown as magenta sticks, and Zn ions as gray spheres.


B Effect of Gli1ZF mutants on the binding affinity to DNA as predicted by in silico alanine scanning . The ∆ ∆ G was calculated along MD trajectories as the difference between the ∆ G of each Gli1ZF alanine mutant and Gli1ZF ‐ WT . Results are shown as ∆ ∆ G values in kcal / mol calculated by means of the MM ‐ PBSA methods ± SEM .

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B Effect of Gli1ZF mutants on the binding affinity to DNA as predicted by in silico alanine scanning. The ∆∆G was calculated along MD trajectories as the difference between the ∆G of each Gli1ZF alanine mutant and Gli1ZF‐WT. Results are shown as ∆∆G values in kcal/mol calculated by means of the MM‐PBSA methods ± SEM.


C Effect of Gli1ZF mutants on Gli1 ‐ dependent transcriptional activation . Luciferase assay was performed in HEK293T cells transfected with 12XGliBS ‐ Luc ( GliBS , Gli binding site ) , pRL ‐ TK Renilla ( normalization control ) , Flag ‐ Gli1 WT or the indicated Flag ‐ Gli1 mutants . Data show the mean ± SD of three independent experiments . * P 0 . 01 ; * * P 0 . 05 versus Gli1 WT . Western blot analysis of Flag ‐ Gli1 WT or the indicated Flag ‐ Gli1 mutant expression levels ( bottom panel ) .

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C Effect of Gli1ZF mutants on Gli1‐dependent transcriptional activation. Luciferase assay was performed in HEK293T cells transfected with 12XGliBS‐Luc (GliBS, Gli binding site), pRL‐TK Renilla (normalization control), Flag‐Gli1 WT or the indicated Flag‐Gli1 mutants. Data show the mean ± SD of three independent experiments. *P 0.01; **P 0.05 versus Gli1 WT. Western blot analysis of Flag‐Gli1 WT or the indicated Flag‐Gli1 mutant expression levels (bottom panel).