The microtubule cytoskeleton is changed in COs upon ECE2 KO . Example images of ac - tub IHC in CTRL vs . ECE2 KO COs ( Scalebar = 100 µm ) .

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The microtubule cytoskeleton is changed in COs upon ECE2 KO. Example images of ac-tub IHC in CTRL vs. ECE2 KO COs (Scalebar = 100 µm).


The microtubule cytoskeleton is changed in COs upon ECE2 KO . Quantification of ac - tub in ventricles of ECE2 KO COs by measurement of the mean grey value in Fiji shows significant reduction upon ECE2 KO ( n = number of analysed ventricles ; * P = 0 . 019 in One Way ANOVA and Tukey Pairwise Multiple Comparison ) .

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The microtubule cytoskeleton is changed in COs upon ECE2 KO. Quantification of ac-tub in ventricles of ECE2 KO COs by measurement of the mean grey value in Fiji shows significant reduction upon ECE2 KO (n=number of analysed ventricles; *P = 0.019 in One Way ANOVA and Tukey Pairwise Multiple Comparison).


Apico - basal polarity is impaired upon ECE2 inhibition and KO as visible in example images of ARL13B IHC in CTRL vs . KO COs ( H ; Scalebar = 100 µm ) . Quantification of germinal zones for normally high ( " 1 " ) vs . reduced ( " 0 " ) apical ARL13B reveals a reduction in the proportion of normal ventricles in the absence of ECE2 ( n = number of analysed ventricles from 2 batches ; * * * * P < 0 . 0001 in exact binomial test ) .

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Apico-basal polarity is impaired upon ECE2 inhibition and KO as visible in example images of ARL13B IHC in CTRL vs. KO COs (H; Scalebar = 100 µm). Quantification of germinal zones for normally high ("1") vs. reduced ("0") apical ARL13B reveals a reduction in the proportion of normal ventricles in the absence of ECE2 (n=number of analysed ventricles from 2 batches; ****P < 0.0001 in exact binomial test).


Analysis of CTRL and ECE2 KO COs 1 dpe with GAP43 - GFP reveals RG that delaminated ( white arrowheads ) and / or lost their bipolar morphology ( red arrowheads ) in the KO COs ( Scalebar = 25 µm ) .

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Analysis of CTRL and ECE2 KO COs 1 dpe with GAP43-GFP reveals RG that delaminated (white arrowheads) and/or lost their bipolar morphology (red arrowheads) in the KO COs (Scalebar = 25 µm).


Binning analysis 1 dpe after forced exression of ECE2 in the developing mouse cortex reveals ectopic positioning of Pax6 + GFP + neural progenitors with significantly reduced localisation to bin2 ( A ) and increased localisation of Ctip2 + deep layer neurons to bin2 with reduction in bin5 ( B ) ( for binning strategy see Figure EV2B ; n = 3 CTRL and 5 ECE2 OX brains E13 - 14 ; data shown as bars with mean ± SEM ; * P < 0 . 05 and * * P < 0 . 01 in Student ' s t - test ) .

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Binning analysis 1 dpe after forced exression of ECE2 in the developing mouse cortex reveals ectopic positioning of Pax6+GFP+ neural progenitors with significantly reduced localisation to bin2 (A) and increased localisation of Ctip2+ deep layer neurons to bin2 with reduction in bin5 (B) (for binning strategy see Figure EV2B; n=3 CTRL and 5 ECE2 OX brains E13-14; data shown as bars with mean±SEM; *P < 0.05 and **P < 0.01 in Student's t-test).


Example images E13 - 14 of IHC for Pax6 and Ctip2 upon CTRL and ECE2 OX conditions ( Scalebar = 100 µm ; electroporated cells shown in green ) . Ventricle marked by V .

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Example images E13-14 of IHC for Pax6 and Ctip2 upon CTRL and ECE2 OX conditions (Scalebar = 100 µm; electroporated cells shown in green). Ventricle marked by V.


E13 - 16 images and analysis of ECE2 OX in the developing mouse cortex reveal ectopic positioning of Sox2 + ( D ) progenitors and Ctip2 + deep - ( D ) and Satb2 + upper layer neurons ( E ) ( Scalebars = 100 µm ; electroporated cells shown in green ) . F , Quantification at 3dpe , counting the proportion of brains with normal vs . abnormal positioning of Sox2 + neural progenitors and Ctip2 + and Satb2 + neurons and that of brains with intact vs . disrupted basal membrane ( n = 5 CTRL and 8 ECE2 OX brains ; exact binomial test : * * * * P < 0 . 0001 ) . Ventricle marked by V .

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E13-16 images and analysis of ECE2 OX in the developing mouse cortex reveal ectopic positioning of Sox2+ (D) progenitors and Ctip2+ deep- (D) and Satb2+ upper layer neurons (E) (Scalebars = 100 µm; electroporated cells shown in green). F, Quantification at 3dpe, counting the proportion of brains with normal vs. abnormal positioning of Sox2+ neural progenitors and Ctip2+ and Satb2+ neurons and that of brains with intact vs. disrupted basal membrane (n=5 CTRL and 8 ECE2 OX brains; exact binomial test: ****P < 0.0001). Ventricle marked by V.


ECE2 OX in COs ( electroporated cells shown in green ; Scalebars = 50 µm ; n = number of CO ventricles analysed from 2 batches ) . G , Example images of COs 7 days after CTRL or ECE2 OX electroporation and IHC for PH3 + mitotic cells and NEUN + neuronal nuclei . H , Quantification of PH3 + cells relative to electroporated apical surface length reveals a reduction upon ECE2 OX in CTRL CO background ( * P = 0 . 046 in Kruskal - Wallis One Way Analysis of Variance on Ranks and Dunn ' s Pairwise Comparison ) . I , Quantification of GFP + neurons as proportion of all GFP + cells reveals an increase upon ECE2 OX in COs ( * P = 0 . 021 in Kruskal - Wallis One Way Analysis of Variance on Ranks and Dunn ' s Pairwise Comparison ) . J , Quantification of ectopic neurons localising to the VZ ' relative to electroporated radial units ( GFP + cells in the VZ ' ) reveals a significant increase upon ECE2 OX ( * * * P < 0 . 001 in Kruskal - Wallis One Way Analysis of Variance on Ranks and Dunn ' s Pairwise Comparison ) . Ventricle - like lumen marked by V ' .

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ECE2 OX in COs (electroporated cells shown in green; Scalebars = 50 µm; n = number of CO ventricles analysed from 2 batches). G, Example images of COs 7 days after CTRL or ECE2 OX electroporation and IHC for PH3+ mitotic cells and NEUN+ neuronal nuclei. H, Quantification of PH3+ cells relative to electroporated apical surface length reveals a reduction upon ECE2 OX in CTRL CO background (*P = 0.046 in Kruskal-Wallis One Way Analysis of Variance on Ranks and Dunn's Pairwise Comparison). I, Quantification of GFP+ neurons as proportion of all GFP+ cells reveals an increase upon ECE2 OX in COs (*P = 0.021 in Kruskal-Wallis One Way Analysis of Variance on Ranks and Dunn's Pairwise Comparison). J, Quantification of ectopic neurons localising to the VZ' relative to electroporated radial units (GFP+ cells in the VZ') reveals a significant increase upon ECE2 OX (***P < 0.001 in Kruskal-Wallis One Way Analysis of Variance on Ranks and Dunn's Pairwise Comparison). Ventricle-like lumen marked by V'.


, Forced expression of ECE2 in ECE2 KO COs rescues neuronal positioning . K , Example images of CTRL and ECE2 electroporated ECE2 KO COs 7 dpe . L , Quantification of ectopic neurons relative to electroporated radial units reveals a decrease upon ECE2 OX in ECE2 KO COs , thus rescueing the neuronal positioning phenotype in the absence of ECE2 OX ( * * P = 0 . 002 in Kruskal - Wallis One Way Analysis of Variance on Ranks and Dunn ' s Pairwise Comparison ) . Ventricle - like lumen marked by V ' .

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, Forced expression of ECE2 in ECE2 KO COs rescues neuronal positioning. K, Example images of CTRL and ECE2 electroporated ECE2 KO COs 7 dpe. L, Quantification of ectopic neurons relative to electroporated radial units reveals a decrease upon ECE2 OX in ECE2 KO COs, thus rescueing the neuronal positioning phenotype in the absence of ECE2 OX (**P = 0.002 in Kruskal-Wallis One Way Analysis of Variance on Ranks and Dunn's Pairwise Comparison). Ventricle-like lumen marked by V'.


Volcano plot visualizes the proteomic analysis from 60d old CO lysates showing proteins with significantly lower and higher expression upon ECE2 KO ( n = 3 batches of 60d old CTRL and ECE2 KO COs ; FDR 0 . 05 ; s0 1 ; dashed lines indicating the cut - off ; names of example proteins are shown ) .

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Volcano plot visualizes the proteomic analysis from 60d old CO lysates showing proteins with significantly lower and higher expression upon ECE2 KO (n=3 batches of 60d old CTRL and ECE2 KO COs; FDR 0.05; s0 1; dashed lines indicating the cut-off; names of example proteins are shown).


Volcano plot of proteomic analysis of cell culture supernatant from 55d old CTRL and ECE2 KO COs shows two significantly downregulated proteins ( red ) in the secretome ( n = 3 batches of CTRL and ECE2 KO COs ; FDR 0 . 05 ; s0 1 ; dashed lines indicating the cut - off ) . Non - significantly changed proteins of interest are highlighted in green below the cut - off curves .

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Volcano plot of proteomic analysis of cell culture supernatant from 55d old CTRL and ECE2 KO COs shows two significantly downregulated proteins (red) in the secretome (n=3 batches of CTRL and ECE2 KO COs; FDR 0.05; s0 1; dashed lines indicating the cut-off). Non-significantly changed proteins of interest are highlighted in green below the cut-off curves.


( B ) Representative emission spectra of mixtures containing ECFP - D4H , mVenus - Lact - C2 , and liposomes containing indicated compositions of lipids . Note that FRET signal was increased in the presence of both cholesterol ( 50 % ) and phosphatidylserine ( DOPS ) ( 20 % ) and that FRET signal was decreased when sphingomyelin ( SM ) ( 25 % ) was additionally incorporated into the liposomes . Asterisks indicate positions of emission maximum of ECFP ( * 477 nm ) and FRET ( * * 525 nm ) . DOPC , phosphatidylcholine ( 1 , 2 - dioleoyl - sn - glycero - 3 - phosphocholine ) ; DOPS , phosphatidylserine ( 1 , 2 - dioleoyl - sn - glycero - 3 - phospho - L - serine ) ; Chol . , cholesterol ; SM , sphingomyelin ( N - oleoyl - D - erythro - sphingosylphosphorylcholine ) . ( C ) Quantification of the ratio of FRET signal at 525 nm to ECFP emission at 480 nm ( FRET / ECFP ) ( see Materials and Methods ) from mixtures as shown in ( D ) ( mean ± SEM , n = 4 independent experiments ; Dunnett ' s multiple comparisons test , * * P < 0 . 0001 ) .

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(B) Representative emission spectra of mixtures containing ECFP-D4H, mVenus-Lact-C2, and liposomes containing indicated compositions of lipids. Note that FRET signal was increased in the presence of both cholesterol (50%) and phosphatidylserine (DOPS) (20%) and that FRET signal was decreased when sphingomyelin (SM) (25%) was additionally incorporated into the liposomes. Asterisks indicate positions of emission maximum of ECFP (*477 nm) and FRET (**525 nm). DOPC, phosphatidylcholine (1,2-dioleoyl-sn-glycero-3-phosphocholine); DOPS, phosphatidylserine (1,2-dioleoyl-sn-glycero-3-phospho-L-serine); Chol., cholesterol; SM, sphingomyelin (N-oleoyl-D-erythro-sphingosylphosphorylcholine). (C) Quantification of the ratio of FRET signal at 525 nm to ECFP emission at 480 nm (FRET/ECFP) (see Materials and Methods) from mixtures as shown in (D) (mean ± SEM, n = 4 independent experiments; Dunnett's multiple comparisons test, **P < 0.0001).


( D ) Left : Time course of normalized mCherry signal , as assessed by TIRF microscopy , from GRAMD1 triple knockout ( TKO ) HeLa cells expressing mCherry - GRAM1b . Cells were pre - incubated with either control imaging buffer ( for control ) or imaging buffer containing purified ECFP - D4H proteins ( 3 µM ) ( for masking accessible PM cholesterol ) for 30 min at 37 ° C and imaged with the same buffer conditions . SMase treatment ( 100 mU / ml ) is indicated . Right : Values of ΔF / F0 corresponding to the end of the experiment as indicated by the arrow [ mean ± SEM , n = 32 cells ( Control ) , n = 35 cells ( Pre - incubation w / ECFP - D4H ) ; data are pooled from two independent experiments for each condition ; two - tailed unpaired Student ' s t - test , * * P < 0 . 0001 ] .

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(D) Left: Time course of normalized mCherry signal, as assessed by TIRF microscopy, from GRAMD1 triple knockout (TKO) HeLa cells expressing mCherry-GRAM1b. Cells were pre-incubated with either control imaging buffer (for control) or imaging buffer containing purified ECFP-D4H proteins (3 µM) (for masking accessible PM cholesterol) for 30 min at 37°C and imaged with the same buffer conditions. SMase treatment (100 mU/ml) is indicated. Right: Values of ΔF/F0 corresponding to the end of the experiment as indicated by the arrow [mean ± SEM, n = 32 cells (Control), n = 35 cells (Pre-incubation w/ ECFP-D4H); data are pooled from two independent experiments for each condition; two-tailed unpaired Student's t-test, **P < 0.0001].


( E ) Left : Time course of normalized EGFP signal , as assessed by TIRF microscopy , from GRAMD1 triple knockout ( TKO ) HeLa cells expressing EGFP - GRAM1b together with either mCherry for control or mCherry - Lact - C2 for PS masking . SMase treatment ( 100 mU / ml ) is indicated . Right : Values of ΔF / F0 corresponding to the end of the experiment as indicated by the arrow [ mean ± SEM , n = 53 cells ( mCherry ) , n = 32 cells ( mCherry - Lact - C2 OE ) ; data are pooled from four independent experiments for each condition ; two - tailed unpaired Student ' s t - test , * * P < 0 . 0001 ] .

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(E) Left: Time course of normalized EGFP signal, as assessed by TIRF microscopy, from GRAMD1 triple knockout (TKO) HeLa cells expressing EGFP-GRAM1b together with either mCherry for control or mCherry-Lact-C2 for PS masking. SMase treatment (100 mU/ml) is indicated. Right: Values of ΔF/F0 corresponding to the end of the experiment as indicated by the arrow [mean ± SEM, n = 53 cells (mCherry), n = 32 cells (mCherry-Lact-C2 OE); data are pooled from four independent experiments for each condition; two-tailed unpaired Student's t-test, **P < 0.0001].


( C - E ) Liposome sedimentation assays of wild - type GRAM1b proteins ( WT ) and mutant versions of GRAM1b proteins carrying indicated mutations ( K161A , R191A , and K161A / R191A ) . Liposomes containing the indicated mole % lipids were incubated with purified GRAM1b proteins as shown . Bound proteins [ pellet , ( P ) ] were separated from the unbound proteins [ supernatant , ( S ) ] , run on SDS - PAGE and visualized by colloidal blue staining ( mean ± SEM , n = 3 independent experiments for all conditions in phosphatidylserine ( PS ) sensitivity , co - incidence detection , and anionic lipid selectivity assays ; n = 12 independent experiments for WT , n = 3 independent experiments for K161A and R191A , and n = 6 independent experiments for K161A / R191A in the cholesterol sensitivity assay ) . DOPC , phosphatidylcholine ( 1 , 2 - dioleoyl - sn - glycero - 3 - phosphocholine ) ; DOPS , phosphatidylserine ( 1 , 2 - dioleoyl - sn - glycero - 3 - phospho - L - serine ) ; Chol . , cholesterol ; PA , phosphatidic acid ( 1 , 2 - dioleoyl - sn - glycero - 3 - phosphate ) ; PI ( 4 ) P , phosphatidylinositol 4 - phosphate ; PI ( 4 , 5 ) P2 , phosphatidylinositol 4 , 5 - bisphosphate ; PI , L - α - phosphatidylinositol .

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(C-E) Liposome sedimentation assays of wild-type GRAM1b proteins (WT) and mutant versions of GRAM1b proteins carrying indicated mutations (K161A, R191A, and K161A/R191A). Liposomes containing the indicated mole% lipids were incubated with purified GRAM1b proteins as shown. Bound proteins [pellet, (P)] were separated from the unbound proteins [supernatant, (S)], run on SDS-PAGE and visualized by colloidal blue staining (mean ± SEM, n = 3 independent experiments for all conditions in phosphatidylserine (PS) sensitivity, co-incidence detection, and anionic lipid selectivity assays; n = 12 independent experiments for WT, n = 3 independent experiments for K161A and R191A, and n = 6 independent experiments for K161A/R191A in the cholesterol sensitivity assay). DOPC, phosphatidylcholine (1,2-dioleoyl-sn-glycero-3-phosphocholine); DOPS, phosphatidylserine (1,2-dioleoyl-sn-glycero-3-phospho-L-serine); Chol., cholesterol; PA, phosphatidic acid (1,2-dioleoyl-sn-glycero-3-phosphate); PI(4)P, phosphatidylinositol 4-phosphate; PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PI, L-α-phosphatidylinositol.


( B ) Sterol specificity of the GRAM domain of GRAMD1b ( GRAM1b ) as assessed via liposome sedimentation assays . Liposomes containing the indicated mole % lipids were incubated with purified GRAM1b proteins as indicated . Bound proteins [ pellet , ( P ) ] were separated from the unbound proteins [ supernatant , ( S ) ] , run on SDS - PAGE and visualized by colloidal blue staining ( mean ± SEM , n = 6 independent experiments for liposomes containing only DOPC , n = 3 independent experiments for the rest ) . DOPC , phosphatidylcholine ( 1 , 2 - dioleoyl - sn - glycero - 3 - phosphocholine ) .

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(B) Sterol specificity of the GRAM domain of GRAMD1b (GRAM1b) as assessed via liposome sedimentation assays. Liposomes containing the indicated mole% lipids were incubated with purified GRAM1b proteins as indicated. Bound proteins [pellet, (P)] were separated from the unbound proteins [supernatant, (S)], run on SDS-PAGE and visualized by colloidal blue staining (mean ± SEM, n = 6 independent experiments for liposomes containing only DOPC, n = 3 independent experiments for the rest). DOPC, phosphatidylcholine (1,2-dioleoyl-sn-glycero-3-phosphocholine).


( D - E ) Liposome sedimentation assays of wild - type GRAM1b proteins ( WT ) and mutant versions of GRAM1b proteins carrying indicated mutations ( R189W , R189W / R191A ) . Liposomes containing the indicated mole % lipids were incubated with purified GRAM1b proteins as shown . Bound proteins [ pellet , ( P ) ] were separated from the unbound proteins [ supernatant , ( S ) ] , run on SDS - PAGE and visualized by colloidal blue staining [ mean ± SEM , n = 3 independent experiments for all conditions in the co - incidence detection assay ; n = 3 independent experiments for R189W and R189W / R191A , n = 6 independent experiments for WT in phosphatidylserine ( PS ) sensitivity assay ; n = 3 independent experiments for R189W and R189W / R191A , n = 5 independent experiments for WT in cholesterol sensitivity assay ] . DOPC , phosphatidylcholine ( 1 , 2 - dioleoyl - sn - glycero - 3 - phosphocholine ) ; DOPS , phosphatidylserine ( 1 , 2 - dioleoyl - sn - glycero - 3 - phospho - L - serine ) ; Chol . , cholesterol .

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

(D-E) Liposome sedimentation assays of wild-type GRAM1b proteins (WT) and mutant versions of GRAM1b proteins carrying indicated mutations (R189W, R189W/R191A). Liposomes containing the indicated mole% lipids were incubated with purified GRAM1b proteins as shown. Bound proteins [pellet, (P)] were separated from the unbound proteins [supernatant, (S)], run on SDS-PAGE and visualized by colloidal blue staining [mean ± SEM, n = 3 independent experiments for all conditions in the co-incidence detection assay; n = 3 independent experiments for R189W and R189W/R191A, n = 6 independent experiments for WT in phosphatidylserine (PS) sensitivity assay; n = 3 independent experiments for R189W and R189W/R191A, n = 5 independent experiments for WT in cholesterol sensitivity assay]. DOPC, phosphatidylcholine (1,2-dioleoyl-sn-glycero-3-phosphocholine); DOPS, phosphatidylserine (1,2-dioleoyl-sn-glycero-3-phospho-L-serine); Chol., cholesterol.


( F ) Confocal images of live GRAMD1 TKO HeLa cells expressing either wild - type ( WT ) or mutant versions of EGFP - tagged GRAM domain of GRAMD1b ( EGFP - GRAM1b ) constructs as indicated . Cells were treated with SMase ( 100 mU / ml for 1 hour at 37 ° C ) before imaging . Insets show at higher magnification the regions indicated by white dashed boxes . Anionic lipid sensing defective mutants ( K161A , R191A , and K161A / R191A ) , a cholesterol sensing defective mutant ( R189W ) , and a cholesterol / anionic lipid sensing defective mutant ( R189W / R191A ) are shown . Note the absence of ( R189W , R189W / R191A , and K161A / R191A ) or significantly reduced ( K161A , R191A ) PM recruitment of the mutant versions of EGFP - GRAM1b compared to the strong PM recruitment of wild - type EGFP - GRAM1b ( WT ) . Scale bars , 10 µm .

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

(F) Confocal images of live GRAMD1 TKO HeLa cells expressing either wild-type (WT) or mutant versions of EGFP-tagged GRAM domain of GRAMD1b (EGFP-GRAM1b) constructs as indicated. Cells were treated with SMase (100 mU/ml for 1 hour at 37°C) before imaging. Insets show at higher magnification the regions indicated by white dashed boxes. Anionic lipid sensing defective mutants (K161A, R191A, and K161A/R191A), a cholesterol sensing defective mutant (R189W), and a cholesterol/anionic lipid sensing defective mutant (R189W/R191A) are shown. Note the absence of (R189W, R189W/R191A, and K161A/R191A) or significantly reduced (K161A, R191A) PM recruitment of the mutant versions of EGFP-GRAM1b compared to the strong PM recruitment of wild-type EGFP-GRAM1b (WT). Scale bars, 10 µm.


( G ) Quantification of the ratio of PM EGFP - GRAM1b signals to the cytosolic EGFP - GRAM1b signals , as assessed by confocal microscopy and line scan analysis from GRAMD1 TKO HeLa cells expressing either wild - type ( WT ) or mutant versions of EGFP - GRAM1b with or without SMase treatment ( 100 mU / ml for 1 hour at 37 ° C ) as shown in ( F ) and Appendix Fig S1B ( mean ± SEM , n = 40 cells for WT , and n = 30 cells for other conditions ; data are pooled from four independent experiments for WT and three independent experiments for the rest ; two - tailed unpaired Student ' s t - test , * * P < 0 . 0001 . n . s . denotes not significant ) .

O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of the ratio of PM EGFP-GRAM1b signals to the cytosolic EGFP-GRAM1b signals, as assessed by confocal microscopy and line scan analysis from GRAMD1 TKO HeLa cells expressing either wild-type (WT) or mutant versions of EGFP-GRAM1b with or without SMase treatment (100 mU/ml for 1 hour at 37°C) as shown in (F) and Appendix Fig S1B (mean ± SEM, n = 40 cells for WT, and n = 30 cells for other conditions; data are pooled from four independent experiments for WT and three independent experiments for the rest; two-tailed unpaired Student's t-test, **P < 0.0001. n.s. denotes not significant).


( H ) Left : Time course of normalized EGFP signal , as assessed by TIRF microscopy , from GRAMD1 TKO HeLa cells expressing either wild - type ( WT ) or mutant versions of EGFP - GRAM1b constructs as indicated . SMase treatment ( 100 mU / ml ) is indicated . Right : Values of ΔF / F0 corresponding to the end of the experiment as indicated by the arrow [ mean ± SEM , n = 126 cells ( WT ) , n = 38 cells ( K161A ) , n = 33 cells ( R191A ) , n = 43 cells ( K161A / R191A ) , n = 46 cells ( R189W ) , n = 44 cells ( R189W / R191A ) ; data are pooled from six independent experiments for WT and two independent experiments for the rest ; Dunnett ' s multiple comparisons test , * * P = 0 . 0002 ( WT vs . K161A ) and * * P < 0 . 0001 for the rest ] .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Left: Time course of normalized EGFP signal, as assessed by TIRF microscopy, from GRAMD1 TKO HeLa cells expressing either wild-type (WT) or mutant versions of EGFP-GRAM1b constructs as indicated. SMase treatment (100 mU/ml) is indicated. Right: Values of ΔF/F0 corresponding to the end of the experiment as indicated by the arrow [mean ± SEM, n = 126 cells (WT), n = 38 cells (K161A), n = 33 cells (R191A), n = 43 cells (K161A/R191A), n = 46 cells (R189W), n = 44 cells (R189W/R191A); data are pooled from six independent experiments for WT and two independent experiments for the rest; Dunnett's multiple comparisons test, **P = 0.0002 (WT vs. K161A) and **P < 0.0001 for the rest].


( B ) Liposome tethering by purified GRAMD1b proteins . Left : Representative time course of liposome tethering , as assessed by turbidity of mixtures containing both LPM and LER together with either wild - type GRAMD1b proteins ( WT ) , mutant GRAMD1b R189W proteins ( R189W ) , mutant GRAMD1b R189W / R191A proteins ( RW / RA ) , or buffer alone as indicated . Turbidity , as assessed by measuring absorbance values at 405 nm , reflects clustering of liposomes due to tethering of LPM and LER . Right : Values of turbidity corresponding to the end of the experiment as indicated by the arrow ( mean ± SEM , n = 6 independent experiments for WT , n = 3 independent experiments for buffer alone , R189W , and RW / RA ; Dunnett ' s multiple comparisons test , * * P < 0 . 0001 )

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 B-MEASURED_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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Liposome tethering by purified GRAMD1b proteins. Left: Representative time course of liposome tethering, as assessed by turbidity of mixtures containing both LPM and LER together with either wild-type GRAMD1b proteins (WT), mutant GRAMD1b R189W proteins (R189W), mutant GRAMD1b R189W/R191A proteins (RW/RA), or buffer alone as indicated. Turbidity, as assessed by measuring absorbance values at 405 nm, reflects clustering of liposomes due to tethering of LPM and LER. Right: Values of turbidity corresponding to the end of the experiment as indicated by the arrow (mean ± SEM, n = 6 independent experiments for WT, n = 3 independent experiments for buffer alone, R189W, and RW/RA; Dunnett's multiple comparisons test, **P < 0.0001)


( C ) DHE transfer between liposomes by purified GRAMD1b proteins . Left : Representative time course of DHE transfer from LPM to LER mediated by either wild - type GRAMD1b proteins ( WT ) , mutant GRAMD1b R189W proteins ( R189W ) , mutant GRAMD1b R189W / R191A proteins ( RW / RA ) , or buffer alone as indicated . Methyl - β - cyclodextrin ( MCD ) at 1 mM was used to determine DHE equilibration . Transfer of DHE from LPM to LER resulted in a decrease in fluorescence resonance energy transfer ( FRET ) between DHE and DNS - PE in LPM . A series of liposomes with different DHE mol % were prepared to plot a calibration curve to convert FRET signals to DHE molecules transferred ( see Appendix Fig S2D and Materials and Methods ) . Right : Values of DHE transfer rate of GRAMD1b ( WT ) , GRAMD1b ( R189W ) , and GRAMD1b ( RW / RA ) , as estimated by the FRET - based lipid transfer assay [ mean ± SEM , n = 6 independent experiments for WT , n = 3 independent experiments for R189W and RW /

O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O 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 B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O 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) DHE transfer between liposomes by purified GRAMD1b proteins. Left: Representative time course of DHE transfer from LPM to LER mediated by either wild-type GRAMD1b proteins (WT), mutant GRAMD1b R189W proteins (R189W), mutant GRAMD1b R189W/R191A proteins (RW/RA), or buffer alone as indicated. Methyl-β-cyclodextrin (MCD) at 1 mM was used to determine DHE equilibration. Transfer of DHE from LPM to LER resulted in a decrease in fluorescence resonance energy transfer (FRET) between DHE and DNS-PE in LPM. A series of liposomes with different DHE mol% were prepared to plot a calibration curve to convert FRET signals to DHE molecules transferred (see Appendix Fig S2D and Materials and Methods). Right: Values of DHE transfer rate of GRAMD1b (WT), GRAMD1b (R189W), and GRAMD1b (RW/RA), as estimated by the FRET-based lipid transfer assay [mean ± SEM, n = 6 independent experiments for WT, n = 3 independent experiments for R189W and RW/RA


( A ) Lysates of wild - type ( control ) and GRAMD1 TKO ( TKO ) HeLa cells that stably expressed either EGFP or EGFP - GRAMD1b constructs as indicated [ wild - type ( WT ) , R189W mutant ( R189W ) , R189W / R191A mutant ( RW / RA ) ] were processed by SDS - PAGE and immunoblotted ( IB ) with anti - GFP , anti - GRAMD1b , and anti - actin antibodies .

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

(A) Lysates of wild-type (control) and GRAMD1 TKO (TKO) HeLa cells that stably expressed either EGFP or EGFP-GRAMD1b constructs as indicated [wild-type (WT), R189W mutant (R189W), R189W/R191A mutant (RW/RA)] were processed by SDS-PAGE and immunoblotted (IB) with anti-GFP, anti-GRAMD1b, and anti-actin antibodies.


( B ) Confocal images of live GRAMD1 TKO HeLa cells stably expressing EGFP - GRAMD1b constructs as indicated that were additionally transfected with an accessible PM cholesterol biosensor , mCherry - tagged GRAM domain of GRAMD1b ( mCherry - GRAM1b ) . Cells were treated with SMase ( 100 mU / ml for 1 hour at 37 ° C ) before imaging . Insets show at higher magnification the regions indicated by white dashed boxes . Note the very weak PM recruitment of mCherry - GRAM1b in cells that stably expressed EGFP - GRAMD1b WT , compared to the strong PM recruitment of mCherry - GRAM1b in cells that stably expressed EGFP - GRAMD1b R189W mutant ( R189W ) or EGFP - GRAMD1b R189W / R191A mutant ( RW / RA ) . Scale bars , 10 µm .

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

(B) Confocal images of live GRAMD1 TKO HeLa cells stably expressing EGFP-GRAMD1b constructs as indicated that were additionally transfected with an accessible PM cholesterol biosensor, mCherry-tagged GRAM domain of GRAMD1b (mCherry-GRAM1b). Cells were treated with SMase (100 mU/ml for 1 hour at 37°C) before imaging. Insets show at higher magnification the regions indicated by white dashed boxes. Note the very weak PM recruitment of mCherry-GRAM1b in cells that stably expressed EGFP-GRAMD1b WT, compared to the strong PM recruitment of mCherry-GRAM1b in cells that stably expressed EGFP-GRAMD1b R189W mutant (R189W) or EGFP-GRAMD1b R189W/R191A mutant (RW/RA). Scale bars, 10 µm.


( C ) Quantification of the ratio of PM mCherry - GRAM1b signals to the cytosolic mCherry - GRAM1b signals , as assessed by confocal microscopy and line scan analysis from GRAMD1 TKO HeLa cells expressing mCherry - GRAM1b with SMase treatment ( 100 mU / ml for 1 hour at 37 ° C ) as shown in ( B ) and Fig EV3B ( mean ± SEM , n = 20 cells for each condition ; data are pooled from two independent experiments ; Dunnett ' s multiple comparisons test , * * P < 0 . 0001 . n . s . denotes not significant ) .

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

(C) Quantification of the ratio of PM mCherry-GRAM1b signals to the cytosolic mCherry-GRAM1b signals, as assessed by confocal microscopy and line scan analysis from GRAMD1 TKO HeLa cells expressing mCherry-GRAM1b with SMase treatment (100 mU/ml for 1 hour at 37°C) as shown in (B) and Fig EV3B (mean ± SEM, n = 20 cells for each condition; data are pooled from two independent experiments; Dunnett's multiple comparisons test, **P < 0.0001. n.s. denotes not significant).


( D ) Left : Time course of normalized mCherry signal , as assessed by TIRF microscopy , from GRAMD1 TKO HeLa cells stably expressing either EGFP or EGFP - GRAMD1b constructs as indicated that were additionally transfected with an accessible PM cholesterol biosensor mCherry - GRAM1b . SMase treatment ( 100 mU / ml ) is indicated . Right : Values of ΔF / F0 corresponding to the end of the experiment as indicated by the arrow [ mean ± SEM , n = 31 cells ( EGFP ) , n = 28 cells ( EGFP - GRAMD1b WT ) , n = 23 cells ( EGFP - GRAMD1b R189W ) , n = 27 cells [ EGFP - GRAMD1b R189W / R191A ( RW / RA ) ] ; data are pooled from two independent experiments for each condition ; Dunnett ' s multiple comparisons test , * * P < 0 . 0001 ( EGFP vs . WT and WT vs . R189W ) , * * P = 0 . 0002 ( WT vs . RW / RA ) ] .

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

(D) Left: Time course of normalized mCherry signal, as assessed by TIRF microscopy, from GRAMD1 TKO HeLa cells stably expressing either EGFP or EGFP-GRAMD1b constructs as indicated that were additionally transfected with an accessible PM cholesterol biosensor mCherry-GRAM1b. SMase treatment (100 mU/ml) is indicated. Right: Values of ΔF/F0 corresponding to the end of the experiment as indicated by the arrow [mean ± SEM, n = 31 cells (EGFP), n = 28 cells (EGFP-GRAMD1b WT), n = 23 cells (EGFP-GRAMD1b R189W), n = 27 cells [EGFP-GRAMD1b R189W/R191A (RW/RA)]; data are pooled from two independent experiments for each condition; Dunnett's multiple comparisons test, **P < 0.0001 (EGFP vs. WT and WT vs. R189W), **P = 0.0002 (WT vs. RW/RA)].


( E ) Wild - type ( control ) and GRAMD1 TKO ( TKO ) HeLa cells that stably expressed either EGFP or EGFP - tagged GRAMD1b constructs as indicated [ wild - type ( WT ) , R189W mutant ( R189W ) , R189W / R191A mutant ( RW / RA ) ] , were cultured in the medium supplemented with 10 % lipoprotein - deficient serum ( LPDS ) and mevastatin ( 50 µM ) for 16 hours and then treated with SMase ( 100 mU / ml ) for 3 hours at 37 ° C . Top : Lysates of the cells were processed for SDS - PAGE and IB with anti - SREBP - 2 and anti - actin antibodies . Arrows indicate precursor ( P ) and cleaved ( C ) forms of SREBP - 2 . Bottom : The response rate was obtained by normalizing the ratio of the band intensity of the cleaved SREBP - 2 over the total band intensity of cleaved and precursor forms of SREBP - 2 from the cells with SMase treatment by the one from the cells without SMase treatment for each condition . Note that the suppression of SREBP - 2 cleavage is attenuated in GRAMD1 TKO HeLa cells compared to wild - type control HeLa cells . Note also the rescue by expression of wild - type EGFP - GRAMD1b ( WT ) but not by mutant versions of EGFP - GRAMD1b [ R189W , R189W / R191A ( RW / RA ) ] [ mean ± SEM , n = 4 lysates ( independent experiments ) for each condition ; Dunnett ' s multiple comparisons test , * * P = 0 . 0035 ( Control + EGFP vs . TKO + EGFP ) , * * P < 0 . 0001 ( TKO + EGFP vs . TKO + WT ) , n . s . denotes not significant ] .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O 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 B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Wild-type (control) and GRAMD1 TKO (TKO) HeLa cells that stably expressed either EGFP or EGFP-tagged GRAMD1b constructs as indicated [wild-type (WT), R189W mutant (R189W), R189W/R191A mutant (RW/RA)], were cultured in the medium supplemented with 10% lipoprotein-deficient serum (LPDS) and mevastatin (50 µM) for 16 hours and then treated with SMase (100 mU/ml) for 3 hours at 37°C. Top: Lysates of the cells were processed for SDS-PAGE and IB with anti-SREBP-2 and anti-actin antibodies. Arrows indicate precursor (P) and cleaved (C) forms of SREBP-2. Bottom: The response rate was obtained by normalizing the ratio of the band intensity of the cleaved SREBP-2 over the total band intensity of cleaved and precursor forms of SREBP-2 from the cells with SMase treatment by the one from the cells without SMase treatment for each condition. Note that the suppression of SREBP-2 cleavage is attenuated in GRAMD1 TKO HeLa cells compared to wild-type control HeLa cells. Note also the rescue by expression of wild-type EGFP-GRAMD1b (WT) but not by mutant versions of EGFP-GRAMD1b [R189W, R189W/R191A (RW/RA)] [mean ± SEM, n = 4 lysates (independent experiments) for each condition; Dunnett's multiple comparisons test, **P = 0.0035 (Control + EGFP vs. TKO + EGFP), **P < 0.0001 (TKO + EGFP vs. TKO + WT), n.s. denotes not significant].


( F ) Amphotericin B resistance of SMase - treated wild - type ( control ) and GRAMD1 TKO ( TKO ) HeLa cells that stably expressed either EGFP or EGFP - tagged GRAMD1b constructs as indicated [ wild - type ( WT ) , R189W mutant ( R189W ) , R189W / R191A mutant ( RW / RA ) ] . Left : Cells that had been pre - treated with SMase ( 100 mU / ml ) for 3 hours at 37 ° C were treated with indicated concentration of Amphotericin B for 20 min at 37ºC . After overnight recovery in culture media , cell viability was measured by detecting ATP present in each well via luminescence ( see Materials and Methods ) . The same number of cells were seeded in each well before SMase treatment . Note the reduced viability of cells with increasing amount of Amphotericin B . Right : Quantification of cell viability with increasing amount of Amphotericin B . Note the resistance of wild - type control and GRAMD1 TKO cells that stably expressed EGFP - GRAMD1b ( WT ) compared to GRAMD1 TKO cells or GRAMD1 TKO cells that stably expressed mutant versions of EGFP - GRAMD1b [ R189W , R189W / R191A ( RW / RA ) ] ( mean ± SEM , n = 3 independent experiments for each condition ) .

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

(F) Amphotericin B resistance of SMase-treated wild-type (control) and GRAMD1 TKO (TKO) HeLa cells that stably expressed either EGFP or EGFP-tagged GRAMD1b constructs as indicated [wild-type (WT), R189W mutant (R189W), R189W/R191A mutant (RW/RA)]. Left: Cells that had been pre-treated with SMase (100 mU/ml) for 3 hours at 37°C were treated with indicated concentration of Amphotericin B for 20 min at 37ºC. After overnight recovery in culture media, cell viability was measured by detecting ATP present in each well via luminescence (see Materials and Methods). The same number of cells were seeded in each well before SMase treatment. Note the reduced viability of cells with increasing amount of Amphotericin B. Right: Quantification of cell viability with increasing amount of Amphotericin B. Note the resistance of wild-type control and GRAMD1 TKO cells that stably expressed EGFP-GRAMD1b (WT) compared to GRAMD1 TKO cells or GRAMD1 TKO cells that stably expressed mutant versions of EGFP-GRAMD1b [R189W, R189W/R191A (RW/RA)] (mean ± SEM, n = 3 independent experiments for each condition).


( B ) Quantification of the ratio of PM signals to the cytosolic signals of wild - type EGFP - GRAM1b ( WT ) and mutant versions of EGFP - GRAM1b ( R166L , Q173I , F182I , T184V , F186L , G187L , A188S ) , as assessed by confocal microscopy and line scan analysis from GRAMD1 TKO HeLa cells , expressing indicated constructs , as shown in ( C ) and Fig EV4A ( mean ± SEM , n = 10 cells for each condition ; data are pooled from one experiment ; Dunnett ' s multiple comparisons test , * * P < 0 . 0001 ) .

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(B) Quantification of the ratio of PM signals to the cytosolic signals of wild-type EGFP-GRAM1b (WT) and mutant versions of EGFP-GRAM1b (R166L, Q173I, F182I, T184V, F186L, G187L, A188S), as assessed by confocal microscopy and line scan analysis from GRAMD1 TKO HeLa cells, expressing indicated constructs, as shown in (C) and Fig EV4A (mean ± SEM, n = 10 cells for each condition; data are pooled from one experiment; Dunnett's multiple comparisons test, **P < 0.0001).


( D ) Confocal images of live wild - type ( control ) and GRAMD1 TKO HeLa cells , expressing either wild - type EGFP - GRAM1b ( WT ) or mutant EGFP - GRAM1b ( G187L ) , with or without SMase treatment ( 100 mU / ml for 1 hour at 37 ° C ) . Insets show at higher magnification the regions indicated by white dashed boxes . Note the recruitment of mutant EGFP - GRAM1b ( G187L ) to the PM even in wild - type HeLa cells at rest , which was further enhanced by SMase treatment . Scale bars , 10 µm .

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(D) Confocal images of live wild-type (control) and GRAMD1 TKO HeLa cells, expressing either wild-type EGFP-GRAM1b (WT) or mutant EGFP-GRAM1b (G187L), with or without SMase treatment (100 mU/ml for 1 hour at 37°C). Insets show at higher magnification the regions indicated by white dashed boxes. Note the recruitment of mutant EGFP-GRAM1b (G187L) to the PM even in wild-type HeLa cells at rest, which was further enhanced by SMase treatment. Scale bars, 10 µm.


( E ) Quantification of the ratio of PM signals to the cytosolic signals of wild - type EGFP - GRAM1b ( WT ) and mutant EGFP - GRAM1b ( G187L ) , as assessed by confocal microscopy and line scan analysis from GRAMD1 TKO HeLa cells , expressing indicated constructs , with or without SMase treatment ( 100 mU / ml for 1 hour at 37 ° C ) , as shown in ( D ) ( mean ± SEM , n = 10 cells for each condition ; data are pooled from one experiment ) .

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(E) Quantification of the ratio of PM signals to the cytosolic signals of wild-type EGFP-GRAM1b (WT) and mutant EGFP-GRAM1b (G187L), as assessed by confocal microscopy and line scan analysis from GRAMD1 TKO HeLa cells, expressing indicated constructs, with or without SMase treatment (100 mU/ml for 1 hour at 37°C), as shown in (D) (mean ± SEM, n = 10 cells for each condition; data are pooled from one experiment).


( F ) A confocal image of live GRAMD1 TKO HeLa cells expressing mutant EGFP - GRAM1b ( G187L ) . Cells were cultured in the medium supplemented with 10 % lipoprotein deficient serum ( LPDS ) and mevastatin ( 50 µM ) for 16 hours to deplete accessible cholesterol before imaging . An inset shows at higher magnification the region indicated by a white dashed box . Note the absence of PM recruitment ( compare to ( D ) ) . Scale bars , 10 µm .

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(F) A confocal image of live GRAMD1 TKO HeLa cells expressing mutant EGFP-GRAM1b (G187L). Cells were cultured in the medium supplemented with 10% lipoprotein deficient serum (LPDS) and mevastatin (50 µM) for 16 hours to deplete accessible cholesterol before imaging. An inset shows at higher magnification the region indicated by a white dashed box. Note the absence of PM recruitment (compare to (D)). Scale bars, 10 µm.


( G ) Time course of normalized EGFP signal , as assessed by TIRF microscopy , from GRAMD1 TKO ( TKO ) HeLa cells expressing either wild - type EGFP - GRAM1b ( WT ) or mutant EGFP - GRAM1b ( G187L ) as indicated . Cells were cultured in the medium supplemented with 10 % lipoprotein deficient serum ( LPDS ) and mevastatin ( 50 µM ) for 16 hours before imaging . SMase treatment ( 100 mU / ml ) is indicated . Right : Values of ΔF / F0 corresponding to the end of the experiment as indicated by the arrow [ mean ± SEM , n = 36 cells ( WT ) , n = 37 cells ( G187L ) , data are pooled from two independent experiments for each condition ; two - tailed unpaired Student ' s t - test , * * P = 0 . 0003 ] . Note the rapid PM recruitment of G187L mutant compared to WT .

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(G) Time course of normalized EGFP signal, as assessed by TIRF microscopy, from GRAMD1 TKO (TKO) HeLa cells expressing either wild-type EGFP-GRAM1b (WT) or mutant EGFP-GRAM1b (G187L) as indicated. Cells were cultured in the medium supplemented with 10% lipoprotein deficient serum (LPDS) and mevastatin (50 µM) for 16 hours before imaging. SMase treatment (100 mU/ml) is indicated. Right: Values of ΔF/F0 corresponding to the end of the experiment as indicated by the arrow [mean ± SEM, n = 36 cells (WT), n = 37 cells (G187L), data are pooled from two independent experiments for each condition; two-tailed unpaired Student's t-test, **P = 0.0003]. Note the rapid PM recruitment of G187L mutant compared to WT.


( H , I ) Liposome sedimentation assays of wild - type GRAM1b ( WT ) and mutant GRAM1b ( G187L ) . Liposomes containing the indicated mole % lipids were incubated with purified GRAM1b proteins as shown . Bound proteins [ pellet , ( P ) ] were separated from the unbound proteins [ supernatant , ( S ) ] , run on SDS - PAGE and visualized by colloidal blue staining [ mean ± SEM , n = 3 independent experiments for all the conditions ; ( H ) Left ( 10 % PS ) : Holm - Sidak t - test for multiple comparisons , * P = 0 . 022679 at 40 % cholesterol , * * P = 0 . 009195 at 50 % cholesterol . Right ( 20 % PS ) : Holm - Sidak t - test for multiple comparisons , * P = 0 . 023163 at 30 % cholesterol , * * P = 0 . 000327 at 40 % cholesterol , * P = 0 . 017194 at 50 % cholesterol . ( I ) Holm - Sidak t - test for multiple comparisons , n . s . denotes not significant , * * P < 0 . 000001 at 50 % DOPC , 50 % cholesterol , * P = 0 . 011271 at 40 % DOPC , 60 % cholesterol ] . DOPC , phosphatidylcholine ( 1 , 2 - dioleoyl - sn - glycero - 3 - phosphocholine ) ; DOPS , phosphatidylserine ( 1 , 2 - dioleoyl - sn - glycero - 3 - phospho - L - serine ) ; Chol . , cholesterol .

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

(H, I) Liposome sedimentation assays of wild-type GRAM1b (WT) and mutant GRAM1b (G187L). Liposomes containing the indicated mole% lipids were incubated with purified GRAM1b proteins as shown. Bound proteins [pellet, (P)] were separated from the unbound proteins [supernatant, (S)], run on SDS-PAGE and visualized by colloidal blue staining [mean ± SEM, n = 3 independent experiments for all the conditions; (H) Left (10% PS): Holm-Sidak t-test for multiple comparisons, *P = 0.022679 at 40% cholesterol, **P = 0.009195 at 50% cholesterol. Right (20% PS): Holm-Sidak t-test for multiple comparisons, *P = 0.023163 at 30% cholesterol, **P = 0.000327 at 40% cholesterol, *P = 0.017194 at 50% cholesterol. (I) Holm-Sidak t-test for multiple comparisons, n.s. denotes not significant, **P < 0.000001 at 50% DOPC, 50% cholesterol, *P = 0.011271 at 40% DOPC, 60% cholesterol]. DOPC, phosphatidylcholine (1,2-dioleoyl-sn-glycero-3-phosphocholine); DOPS, phosphatidylserine (1,2-dioleoyl-sn-glycero-3-phospho-L-serine); Chol., cholesterol.


( A ) Effects of mutation of G187 on the property of the GRAM domain of GRAMD1b ( GRAM1b ) to sense transient expansion of the accessible pool of PM cholesterol . Indicated EGFP - tagged mutant versions of GRAM1b were expressed in GRAMD1 TKO HeLa cells and imaged under confocal microscopy with or without SMase treatment ( 100 mU / ml for 1 hour at 37 ° C ) or with SMase treatment followed by 5 min treatment with Methyl - β - cyclodextrin ( MCD ) ( 5 mM ) to assess their PM recruitment . The mean values ( n = 20 cells from two independent experiments for untreated and SMase - treated conditions ; n = 10 cells from one experiment for SMase & MCD - treated conditions ) of the ratio of PM signals to the cytosolic signals , as assessed by line scan analysis , are presented for each condition as a heatmap . Amino acids are ranked according to Goldman , Engelman and Steitz ( GES ) hydrophobicity scale with the most hydrophobic amino acid on the top .

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

(A) Effects of mutation of G187 on the property of the GRAM domain of GRAMD1b (GRAM1b) to sense transient expansion of the accessible pool of PM cholesterol. Indicated EGFP-tagged mutant versions of GRAM1b were expressed in GRAMD1 TKO HeLa cells and imaged under confocal microscopy with or without SMase treatment (100 mU/ml for 1 hour at 37°C) or with SMase treatment followed by 5 min treatment with Methyl-β-cyclodextrin (MCD) (5 mM) to assess their PM recruitment. The mean values (n = 20 cells from two independent experiments for untreated and SMase-treated conditions; n = 10 cells from one experiment for SMase & MCD-treated conditions) of the ratio of PM signals to the cytosolic signals, as assessed by line scan analysis, are presented for each condition as a heatmap. Amino acids are ranked according to Goldman, Engelman and Steitz (GES) hydrophobicity scale with the most hydrophobic amino acid on the top.


( B ) Liposome tethering by purified GRAMD1b proteins . Left : Representative time course of liposome tethering , as assessed by turbidity of mixtures containing both LPM and LER together with either wild - type GRAMD1b proteins ( WT ) , mutant GRAMD1b G187L proteins ( G187L ) , or buffer alone as indicated . Experiments and data analysis were performed as in Fig 4 . Right : Values of turbidity corresponding to the end of the experiment as indicated by the arrow ( mean ± SEM , n = 3 independent experiments ; Dunnett ' s multiple comparisons test , * P = 0 . 0143 ) .

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(B) Liposome tethering by purified GRAMD1b proteins. Left: Representative time course of liposome tethering, as assessed by turbidity of mixtures containing both LPM and LER together with either wild-type GRAMD1b proteins (WT), mutant GRAMD1b G187L proteins (G187L), or buffer alone as indicated. Experiments and data analysis were performed as in Fig 4. Right: Values of turbidity corresponding to the end of the experiment as indicated by the arrow (mean ± SEM, n = 3 independent experiments; Dunnett's multiple comparisons test, *P = 0.0143).


( C ) DHE transfer between liposomes by purified GRAMD1b proteins . Left : Representative time course of DHE transfer from LPM to LER mediated by either wild - type GRAMD1b proteins ( WT ) , mutant GRAMD1b G187L proteins ( G187L ) , or buffer alone as indicated . Methyl - β - cyclodextrin ( MCD ) at 1 mM was used to determine DHE equilibration . Experiments and data analysis were performed as in Fig 4 . Right : Values of DHE transfer rate of wild - type GRAMD1b ( WT ) and mutant GRAMD1b ( G187L ) , as estimated by the FRET - based lipid transfer assay ( mean ± SEM , n = 3 independent experiments ; two - tailed unpaired Student ' s t - test , * * P < 0 . 0014 ) .

O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O 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-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 B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O 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) DHE transfer between liposomes by purified GRAMD1b proteins. Left: Representative time course of DHE transfer from LPM to LER mediated by either wild-type GRAMD1b proteins (WT), mutant GRAMD1b G187L proteins (G187L), or buffer alone as indicated. Methyl-β-cyclodextrin (MCD) at 1 mM was used to determine DHE equilibration. Experiments and data analysis were performed as in Fig 4. Right: Values of DHE transfer rate of wild-type GRAMD1b (WT) and mutant GRAMD1b (G187L), as estimated by the FRET-based lipid transfer assay (mean ± SEM, n = 3 independent experiments; two-tailed unpaired Student's t-test, **P < 0.0014).


( D ) Confocal images of live GRAMD1 TKO HeLa cells that stably expressed either EGFP control or EGFP - GRAMD1b constructs as indicated . Cells were stained with recombinant mCherry - D4H proteins ( 10 mg / ml ) ( accessible PM cholesterol biosensor ) for 15 min at room temperature before imaging . Insets show at higher magnification the regions indicated by white dashed boxes . Note that expression of EGFP - GRAMD1b - WT , but not EGFP - GRAMD1b - R189W , reduced binding of mCherry - D4H to the PM , in GRAMD1 TKO HeLa cells . Further reduction in mCherry - D4H binding to the PM was observed in GRAMD1 TKO HeLa cells that stably expressed EGFP - GRAMD1b - G187L compared to GRAMD1 TKO HeLa cells that stably expressed EGFP - GRAMD1b - WT . Scale bars , 10 µm . ( E ) Values of mCherry - D4H signals at the PM after background subtraction , as assessed by confocal microscopy and line scan analysis of GRAMD1 TKO HeLa cells that stably expressed either EGFP or indicated EGFP - GRAMD1b constructs as shown in ( D ) [ mean ± SEM , n = 30 cells for all the conditions ; data are pooled from three independent experiments ; Dunnett ' s multiple comparisons test , * * P < 0 . 0001 ] .

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(D) Confocal images of live GRAMD1 TKO HeLa cells that stably expressed either EGFP control or EGFP-GRAMD1b constructs as indicated. Cells were stained with recombinant mCherry-D4H proteins (10 mg/ml) (accessible PM cholesterol biosensor) for 15 min at room temperature before imaging. Insets show at higher magnification the regions indicated by white dashed boxes. Note that expression of EGFP-GRAMD1b-WT, but not EGFP-GRAMD1b-R189W, reduced binding of mCherry-D4H to the PM, in GRAMD1 TKO HeLa cells. Further reduction in mCherry-D4H binding to the PM was observed in GRAMD1 TKO HeLa cells that stably expressed EGFP-GRAMD1b-G187L compared to GRAMD1 TKO HeLa cells that stably expressed EGFP-GRAMD1b-WT. Scale bars, 10 µm. (E) Values of mCherry-D4H signals at the PM after background subtraction, as assessed by confocal microscopy and line scan analysis of GRAMD1 TKO HeLa cells that stably expressed either EGFP or indicated EGFP-GRAMD1b constructs as shown in (D) [mean ± SEM, n = 30 cells for all the conditions; data are pooled from three independent experiments; Dunnett's multiple comparisons test, **P < 0.0001].


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 .

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(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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 ) .

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

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 .

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

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 ) .

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

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 .

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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 .

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

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 .

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. 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 .

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

(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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 .

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

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 .

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. 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 .

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

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 ) .

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

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 .

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

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

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 ) .

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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 .

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(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 B-MEASURED_VAR O B-MEASURED_VAR 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

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

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