( 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) .

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

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

(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 ) .

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

(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 .

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

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

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

(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 ] .

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

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

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

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

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

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 .

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

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

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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 ) .

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

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 .

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

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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 ) .

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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 .

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

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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 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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 Interaction of Atg18 with retromer subunits . Genomically tagged yomCherry - fusions of each retromer subunit were expressed in SEY6210 atg18 ∆ , atg21 ∆ , hsv2 ∆ cells , together with a plasmid expressing Atg18HA3yEGFP . Cell lysates were subjected to immunoprecipitation using RFP - Trap magnetic beads and analyzed by SDS - PAGE and Western blotting against the indicated proteins .

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B Interaction of Atg18 with retromer subunits. Genomically tagged yomCherry-fusions of each retromer subunit were expressed in SEY6210 atg18∆, atg21∆, hsv2∆ cells, together with a plasmid expressing Atg18HA3yEGFP. Cell lysates were subjected to immunoprecipitation using RFP-Trap magnetic beads and analyzed by SDS-PAGE and Western blotting against the indicated proteins.


D Atg18 - Vps35 interaction . Genomically tagged Vps35yomCherry was pulled down from lysates of SEY6210 WT or SEY6210 vps26 ∆ strains carrying genomically tagged Atg18HA3yEGFP . Adsorbed proteins were analyzed by SDS - PAGE and Western blotting using the antibodies indicated in brackets . E Signals from the blots in d were quantified on an infrared fluorescence scanner and normalized to the amount of pulled - down Vps35yomCherry . n = 3 independent experiments were analyzed using an unpaired Student ' s t - test . Bars represent the mean and errors bars the SEM . * * * P < 0 . 001 .

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D Atg18-Vps35 interaction. Genomically tagged Vps35yomCherry was pulled down from lysates of SEY6210 WT or SEY6210 vps26∆ strains carrying genomically tagged Atg18HA3yEGFP. Adsorbed proteins were analyzed by SDS-PAGE and Western blotting using the antibodies indicated in brackets. E Signals from the blots in d were quantified on an infrared fluorescence scanner and normalized to the amount of pulled-down Vps35yomCherry. n=3 independent experiments were analyzed using an unpaired Student's t-test. Bars represent the mean and errors bars the SEM. *** P < 0.001.


F Atg18 - Vps26 interaction . Genomically tagged Vps26yomCherry was pulled down from lysates of SEY6210 WT , SEY6210 vps35 ∆ or SEY6210 vps29 ∆ strains carrying genomically tagged Atg18HA3yEGFP . Adsorbed proteins were analyzed as in D . G Proteins from F were quantified as in E . n = 3 independent experiments were analyzed using an unpaired Student ' s t - test . Bars represent the mean and errors bars the SEM . * * * p < 0 . 001 ) .

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F Atg18-Vps26 interaction. Genomically tagged Vps26yomCherry was pulled down from lysates of SEY6210 WT, SEY6210 vps35∆ or SEY6210 vps29∆ strains carrying genomically tagged Atg18HA3yEGFP. Adsorbed proteins were analyzed as in D. G Proteins from F were quantified as in E. n=3 independent experiments were analyzed using an unpaired Student's t-test. Bars represent the mean and errors bars the SEM. *** p < 0.001).


B Salt - induced vacuole fission . The indicated cells were logarithmically grown in YPD and stained with FM4 - 64 . Vacuole morphology was imaged as in A , before and after a mild salt shock with 0 . 5 M of NaCl for 15 min . The look - up table has been inverted to allow better representation of the clusters of extremely small vacuolar fragments in the SNX mutants . Scale bar : 5 µm . C The number of vacuoles per cell was quantified for the samples from B . n = 3 experiments with at least 100 cells per condition were evaluated ; Error bars represent the SEM .

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B Salt-induced vacuole fission. The indicated cells were logarithmically grown in YPD and stained with FM4-64. Vacuole morphology was imaged as in A, before and after a mild salt shock with 0.5 M of NaCl for 15 min. The look-up table has been inverted to allow better representation of the clusters of extremely small vacuolar fragments in the SNX mutants. Scale bar: 5 µm. C The number of vacuoles per cell was quantified for the samples from B. n=3 experiments with at least 100 cells per condition were evaluated; Error bars represent the SEM.


A Vps17 labilizes the Atg18 - Vps26 interaction . Wildtype or vps17 ∆ cells expressing ATG18HA3 - yEGFP from a centromeric plasmid were logarithmically grown in YPD . Genomically tagged Vps26yomCherry was pulled down from whole - cell extracts and analyzed for associated Atg18HA3 - yEGFP by SDS - PAGE and Western blotting . Glucose - 6 - phosphate dehydrogenase ( Zwf1 ) serves as a loading control . The intensity of the interacting Atg18HA3 - yEGFP was quantified on a LICOR fluorescence imager and normalized to the amount of Vps26yomCherry . Values of the wildtype interaction were used as the reference and set to 1 . n = 4 independent experiments were analyzed using an unpaired Student ' s t - test . Bars represent the mean and errors bars the SEM , * * p < 0 . 01 )

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A Vps17 labilizes the Atg18-Vps26 interaction. Wildtype or vps17∆ cells expressing ATG18HA3-yEGFP from a centromeric plasmid were logarithmically grown in YPD. Genomically tagged Vps26yomCherry was pulled down from whole-cell extracts and analyzed for associated Atg18HA3-yEGFP by SDS-PAGE and Western blotting. Glucose-6-phosphate dehydrogenase (Zwf1) serves as a loading control. The intensity of the interacting Atg18HA3-yEGFP was quantified on a LICOR fluorescence imager and normalized to the amount of Vps26yomCherry. Values of the wildtype interaction were used as the reference and set to 1. n = 4 independent experiments were analyzed using an unpaired Student's t-test. Bars represent the mean and errors bars the SEM, ** p < 0.01)


B Epistasis of ATG18 and retromer genes concerning vacuolar morphology . The indicated cells were logarithmically grown in YPD medium , stained with FM4 - 64 and calcofluor white C Quantification of vacuole morphology . The number of vacuoles per cell was measured in the cells from B . The graph shows the fractions of cells displaying the indicated numbers of vacuolar vesicles ( n = 3 biological experiments with at least 100 cells per condition and experiment were analyzed using an unpaired Student ' s t - test . Bars represent the mean and errors bars the SEM . * * p < 0 .

B Epistasis of ATG18 and retromer genes concerning vacuolar morphology. The indicated cells were logarithmically grown in YPD medium, stained with FM4-64 and calcofluor white C Quantification of vacuole morphology. The number of vacuoles per cell was measured in the cells from B. The graph shows the fractions of cells displaying the indicated numbers of vacuolar vesicles (n = 3 biological experiments with at least 100 cells per condition and experiment were analyzed using an unpaired Student's t-test. Bars represent the mean and errors bars the SEM. ** p < 0.01


B Purified Atg18 and CSC - GFP were mixed in a 1 : 1 ratio , incubated together in PBS and analyzed by native PAGE and Western blotting using the antibodies indicated in brackets .

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B Purified Atg18 and CSC-GFP were mixed in a 1:1 ratio, incubated together in PBS and analyzed by native PAGE and Western blotting using the antibodies indicated in brackets.


C Pull - down . Cells ( SEY6210 atg18 ∆ , atg21 ∆ ) expressing genomically tagged Vps26yomCherry and the indicated Atg18HA3 - yEGFP variants were logarithmically grown in SC - URA media . Vps26yomCherry was pulled down from whole - cell extracts with RFP - trap magnetic beads and analyzed by SDS - PAGE and Western blotting against the indicated proteins . Bands were quantified on a LICOR fluorescence imager . Signals of Atg18HA3yEGFP were normalized relative to those of Vps26yomCherry . Vps1 served as a loading control . n = 3 independent replicates . Data were subjected to an unpaired t - test . Bars represent the mean and errors bars the SEM , * * * P < 0 . 001 ) .

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C Pull-down. Cells (SEY6210 atg18∆, atg21∆) expressing genomically tagged Vps26yomCherry and the indicated Atg18HA3-yEGFP variants were logarithmically grown in SC-URA media. Vps26yomCherry was pulled down from whole-cell extracts with RFP-trap magnetic beads and analyzed by SDS-PAGE and Western blotting against the indicated proteins. Bands were quantified on a LICOR fluorescence imager. Signals of Atg18HA3yEGFP were normalized relative to those of Vps26yomCherry. Vps1 served as a loading control. n = 3 independent replicates. Data were subjected to an unpaired t-test. Bars represent the mean and errors bars the SEM, *** P < 0.001).


A , Effect of Atg18T56E on salt - induced vacuole fission . Cells expressing Atg18WTHA3 - yEGFP or Atg18T56E - HA3 - yEGFP from centromeric plasmids in a SEY6210 atg18 ∆ , atg21 ∆ strain were logarithmically grown in SD - URA medium . They were stained and imaged before and after the induction of vacuole fission by a short salt shock Calcofluor white - stained cell walls are only represented in the merge ( blue ) . Scale bar : 5 µm . B , Quantification of the number of vacuoles per cell from A , n = 3 biological experiments with at least 100 cells per condition were scored ; bars represent the mean and error bars the SEM . C , Epistasis of atg18T56E over a vps17 ∆ mutation . The indicated variants of Atg18 - HA3 - yEGFP were expressed from plasmids in a SEY6210 atg18 ∆ , atg21 ∆ , vps17 ∆ strain . Cells were logarithmically grown in SD - URA and imaged Scale bar : 5 µm . D , Quantification of the experiments from C , n = 3 independent experiments with at least 100 cells per condition scored ; bars represent the mean and error bars the SEM .

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A, Effect of Atg18T56E on salt-induced vacuole fission. Cells expressing Atg18WTHA3-yEGFP or Atg18T56E-HA3-yEGFP from centromeric plasmids in a SEY6210 atg18∆, atg21∆ strain were logarithmically grown in SD-URA medium. They were stained and imaged before and after the induction of vacuole fission by a short salt shock Calcofluor white-stained cell walls are only represented in the merge (blue). Scale bar: 5 µm. B, Quantification of the number of vacuoles per cell from A, n = 3 biological experiments with at least 100 cells per condition were scored; bars represent the mean and error bars the SEM. C, Epistasis of atg18T56E over a vps17∆ mutation. The indicated variants of Atg18-HA3-yEGFP were expressed from plasmids in a SEY6210 atg18∆, atg21∆, vps17∆ strain. Cells were logarithmically grown in SD-URA and imaged Scale bar: 5 µm. D, Quantification of the experiments from C, n = 3 independent experiments with at least 100 cells per condition scored; bars represent the mean and error bars the SEM .


A . Atg18 - dependent recruitment of CSC - GFP to small liposomes . SUVs were incubated ( 10 min , 25 ° C ) with purified CSC ( 1 . 5 µM ) alone or in combination with the indicated recombinant Atg18 variants ( 1 . 5 µM ) . The vesicles were sedimented by centrifugation and supernatants ( Sup . ) and pellets were analyzed by SDS - PAGE and Coomassie staining . B . Quantification of the Coomassie signals for Vps35 by densitometry on a LICOR Odyssey scanner . n = 3 independent experiments . Bars represent the mean and error bars the SEM , * * p < 0 . 01 ; * p < 0 . 05 . C . Quantification as in B , but for the Atg18 variants .

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A. Atg18-dependent recruitment of CSC-GFP to small liposomes. SUVs were incubated (10 min, 25°C) with purified CSC (1.5 µM) alone or in combination with the indicated recombinant Atg18 variants (1.5 µM). The vesicles were sedimented by centrifugation and supernatants (Sup.) and pellets were analyzed by SDS-PAGE and Coomassie staining. B. Quantification of the Coomassie signals for Vps35 by densitometry on a LICOR Odyssey scanner. n = 3 independent experiments. Bars represent the mean and error bars the SEM, ** p < 0.01; * p < 0.05. C. Quantification as in B, but for the Atg18 variants.


A Colocalization of WIPI1 with Vps26A . The indicated WIPI1mCherry variants and VPS26EGFP were expressed for 18 h in HK2 cells , from which endogenous WIPI1 had been deleted ( WIPI1 - KO ) . The cells were analyzed by confocal microscopy . Scale bars : 10 μm . Insets show enlargements of the outlined areas . B Colocalization between WIPI1 variants and Vps26 was quantified in the cells from A using the Manders ' colocalization coefficient M2 . The red line indicates the mean ; n = 120 cells per condition , pooled from three independent experiments . P - values were calculated by t - test ( analysis performed with 99 % confidence * * * p < 0 . 0001 ) . C Expression levels of WIPI1mCherry variants . Lysates ( 50 μg of protein per sample ) from the cells in A were analyzed by SDS - PAGE and Western blot against WIPI1 and tubulin .

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A Colocalization of WIPI1 with Vps26A. The indicated WIPI1mCherry variants and VPS26EGFP were expressed for 18 h in HK2 cells, from which endogenous WIPI1 had been deleted (WIPI1-KO). The cells were analyzed by confocal microscopy. Scale bars: 10 μm. Insets show enlargements of the outlined areas. B Colocalization between WIPI1 variants and Vps26 was quantified in the cells from A using the Manders' colocalization coefficient M2. The red line indicates the mean; n = 120 cells per condition, pooled from three independent experiments. P-values were calculated by t-test (analysis performed with 99% confidence ***p < 0.0001). C Expression levels of WIPI1mCherry variants. Lysates (50 μg of protein per sample) from the cells in A were analyzed by SDS-PAGE and Western blot against WIPI1 and tubulin.


D Depletion of VPS35 . HK2 cells expressing the indicated WIPI1eGFP variants were transfected with siRNA against VPS35 ( VPS35 KD ) or a control siRNA pool . Lysates ( 50 μg per sample ) from the cells were analyzed by SDS - PAGE and Western blot against Vps35 and tubulin . E Blots from D were quantified on a LICOR Odyssey fluorescence imager . n = 3 . Red lines indicate the means , n = 3 independent experiments , using a Welch ' s t - test statistical analysis . Bars represent the mean and error bars the SD .

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D Depletion of VPS35. HK2 cells expressing the indicated WIPI1eGFP variants were transfected with siRNA against VPS35 (VPS35 KD) or a control siRNA pool. Lysates (50 μg per sample) from the cells were analyzed by SDS-PAGE and Western blot against Vps35 and tubulin. E Blots from D were quantified on a LICOR Odyssey fluorescence imager. n=3. Red lines indicate the means, n = 3 independent experiments, using a Welch's t-test statistical analysis. Bars represent the mean and error bars the SD.


F Cells from D were analyzed by confocal microscopy 18 h after transfection . Scale bar : 10 μm . G Quantification of tubule length in the cells from F . Data are means ± SD . of n = 210 tubules per condition , from three independent experiments . p values were calculated by unpaired Student ' s t - test . 99 % confidence : * * * = p < 0 . 0001 .

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F Cells from D were analyzed by confocal microscopy 18 h after transfection. Scale bar: 10 μm. G Quantification of tubule length in the cells from F. Data are means ± SD. of n=210 tubules per condition, from three independent experiments. p values were calculated by unpaired Student's t-test. 99% confidence: *** = p < 0.0001.


A , Tf recycling . WIPI1 - KO cells were transfected with WIPI1WT - eGFP , WIPI1S69E - eGFP or WIPI1S69A - eGFP for 18h . Then , they were serum - starved for 1 h , loaded with Alexa Fluor 568 - conjugated Tf , chased at 37 ° C for 1 h without labeled Tf , and analyzed by confocal microscopy . Scale bar : 10 μm . White dashed lines delineate the circumference of the cells . B , Quantification of Tf - fluorescence in the cells from a that expressed WIPI1 variants . Total cell fluorescence was integrated and corrected for background fluorescence . Mean values ± SD are shown . n = 3 independent experiments with a total of 150 cells analyzed per condition . P values were calculated by unpaired Student ' s t - test . 99 % confidence : * * * = p < 0 . 0001 .

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A, Tf recycling. WIPI1-KO cells were transfected with WIPI1WT-eGFP, WIPI1S69E-eGFP or WIPI1S69A-eGFP for 18h. Then, they were serum-starved for 1 h, loaded with Alexa Fluor 568-conjugated Tf, chased at 37°C for 1 h without labeled Tf, and analyzed by confocal microscopy. Scale bar: 10 μm. White dashed lines delineate the circumference of the cells. B, Quantification of Tf-fluorescence in the cells from a that expressed WIPI1 variants. Total cell fluorescence was integrated and corrected for background fluorescence. Mean values ± SD are shown. n = 3 independent experiments with a total of 150 cells analyzed per condition. P values were calculated by unpaired Student's t-test. 99% confidence: *** = p < 0.0001.