Figure 1A - B HT ‑ 29 cells were transfected with non - silencing control siRNA ( sictr ) or siRNAs against USP22 ( siUSP22 ) for 48 h at 20 nM . After transfection , cells were treated with 20 µM zVAD . fmk , 0 . 5 µM BV6 and 1 ng / ml TNFα either for 6 h and analyzed by Western blotting ( A ) or for 18 h , and the percentage of PI - positive cells was assessed by fluorescence - based PI staining ( B ) . Vinculin served as a loading control . C HT ‑ 29 CRISPR / Cas9 control ( ctr ) and USP22 KO cells were analyzed by Western blotting for USP22 expression . GAPDH served as loading control . D HT ‑ 29 control and USP22 KO cells were treated with 20 µM zVAD . fmk , 0 . 5 µM BV6 and 1 ng / ml TNFα after pre - incubation with 30 µM Nec1 - s and 20 µM GSK ' 872 for 1 h and incubated for 18 h before fluorescence - based quantification of PI - positive cells . E HT ‑ 29 control and USP22 KO cells , generated with 2 guide RNAs ( 2g ) , expressing empty vector ( EV ) or PAM mutated 3xFLAG - HA - USP22 WT ( USP22 PAM ) , C185S ( USP22 PAM C185S ) or C185A ( USP22 PAM C185A ) were analyzed by Western blotting for USP22 expression levels . β - Actin was used as a loading control . F HT - 29 control and USP22 KO cells , generated with 2 guide RNAs ( 2g ) , expressing empty vector ( EV ) or PAM mutated 3xFLAG - HA - USP22 WT ( USP22 PAM ) , C185S ( USP22 PAM C185S ) or C185A ( USP22 PAM C185A ) were stimulated with 20 µM zVAD . fmk , 0 . 5 µM BV6 , 1 ng / ml TNFα for 18 h . The percentage of PI - positive cells was assessed by fluorescence - based PI staining .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A-B HT‑29 cells were transfected with non-silencing control siRNA (sictr) or siRNAs against USP22 (siUSP22) for 48 h at 20 nM. After transfection, cells were treated with 20 µM zVAD.fmk, 0.5 µM BV6 and 1 ng/ml TNFα either for 6 h and analyzed by Western blotting (A) or for 18 h, and the percentage of PI-positive cells was assessed by fluorescence-based PI staining (B). Vinculin served as a loading control.C HT‑29 CRISPR/Cas9 control (ctr) and USP22 KO cells were analyzed by Western blotting for USP22 expression. GAPDH served as loading control.D HT‑29 control and USP22 KO cells were treated with 20 µM zVAD.fmk, 0.5 µM BV6 and 1 ng/ml TNFα after pre-incubation with 30 µM Nec1-s and 20 µM GSK'872 for 1 h and incubated for 18 h before fluorescence-based quantification of PI-positive cells.E HT‑29 control and USP22 KO cells, generated with 2 guide RNAs (2g), expressing empty vector (EV) or PAM mutated 3xFLAG-HA-USP22 WT (USP22 PAM), C185S (USP22 PAM C185S) or C185A (USP22 PAM C185A) were analyzed by Western blotting for USP22 expression levels. β-Actin was used as a loading control.F HT-29 control and USP22 KO cells, generated with 2 guide RNAs (2g), expressing empty vector (EV) or PAM mutated 3xFLAG-HA-USP22 WT (USP22 PAM), C185S (USP22 PAM C185S) or C185A (USP22 PAM C185A) were stimulated with 20 µM zVAD.fmk, 0.5 µM BV6, 1 ng/ml TNFα for 18 h. The percentage of PI-positive cells was assessed by fluorescence-based PI staining.


Figure 2A HT ‑ 29 control and USP22 KO cells were stimulated with 20 µM zVAD . fmk , 0 . 5 µM BV6 and 1 ng / ml TNFα for the indicated time points . Detection of indicated proteins was carried out by Western blotting . GAPDH served as a loading control . B HT ‑ 29 control and USP22 KO cells were stimulated with 20 µM zVAD . fmk , 0 . 5 µM BV6 and 1 ng / ml TNFα for 4 h . Detection of indicated proteins was carried out by Western blotting . β - Actin served as a loading control . C HT ‑ 29 control and USP22 KO cells were incubated with 30 µM Nec1 - s or 20 µM Dab for 18 h , as indicated . Cell were stimulated with 20 µM zVAD . fmk , 0 . 5 µM BV6 and 1 ng / ml TNFα for 5 h . 100 μg of each lysate were incubated with 400 U / μl λ - phosphatase for 30 min at 30 ° C . Protein expression of RIPK3 was monitored by Western blotting . β - Actin was used as loading control . High molecular weight RIPK3 ' smears ' were quantified after λ - phosphatase treatment and normalized to total RIPK3 and β - Actin levels .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A HT‑29 control and USP22 KO cells were stimulated with 20 µM zVAD.fmk, 0.5 µM BV6 and 1 ng/ml TNFα for the indicated time points. Detection of indicated proteins was carried out by Western blotting. GAPDH served as a loading control.B HT‑29 control and USP22 KO cells were stimulated with 20 µM zVAD.fmk, 0.5 µM BV6 and 1 ng/ml TNFα for 4 h. Detection of indicated proteins was carried out by Western blotting. β-Actin served as a loading control.C HT‑29 control and USP22 KO cells were incubated with 30 µM Nec1-s or 20 µM Dab for 18 h, as indicated. Cell were stimulated with 20 µM zVAD.fmk, 0.5 µM BV6 and 1 ng/ml TNFα for 5 h. 100 μg of each lysate were incubated with 400 U/μl λ-phosphatase for 30 min at 30 °C. Protein expression of RIPK3 was monitored by Western blotting. β-Actin was used as loading control. High molecular weight RIPK3 'smears' were quantified after λ-phosphatase treatment and normalized to total RIPK3 and β-Actin levels.


Figure 3A HeLa TRex RIPK3 CRISPR / Cas9 control ( ctr ) and USP22 KO cells were treated with 1 µg / ml Dox overnight . Protein expression of induced Strep - RIPK3 was analyzed by Western blotting . GAPDH served as loading control . The asterisk marks an unspecific band . B HeLa TRex RIPK3 control and USP22 KO cells were incubated with 1 µg / ml Dox for 18 h before pre - treatment with 20 µM zVAD . fmk , 5 µM BV6 for 1 h . After pre - treatment , 10 ng / ml TNFα was added and cell death was measured after 4 and 5 h by analysis of PI - positive nuclei . C HeLa TRex RIPK3 control and USP22 KO cells were pre - treated with 20 µM zVAD . fmk , 5 µM BV6 for 1 h . After pre - treatment , 10 ng / ml TNFα were added for 1 , 2 , 3 , 4 and 5 h . Protein expression of phosphorylated RIPK1 , total RIPK1 , total RIPK3 , phosphorylated MLKL , total MLKL and USP22 , without ( left ) or with ( right ) 1 µg / ml Dox treatment overnight , was monitored by Western blotting . GAPDH was used as a loading control . D HT - 29 control cells and RIPK3 KO cells re - expressing PAM - mutated Dox - inducible RIPK3 WT were incubated overnight with 1 µg / ml Dox . Cells were pre - treated with 20 µM zVAD . fmk , 5 µM BV6 for 1 h . After pre - treatment , 10 ng / ml TNFα were added for 2 h , as indicated . Strep - RIPK3 was immunoprecipitated using anti ‑ Strep - beads and the indicated co - immunoprecipitated proteins were analyzed by Western blotting . β - Actin served as a loading control . E USP22 KO HT - 29 cells and USP22 KO cells re - expressing PAM - mutated 3xFLAG - HA - USP22 were pre - treated with 20 µM zVAD . fmk , 5 µM BV6 for 1 h . After pre - treatment , 10 ng / ml TNFα was added for 2 h , as indicated . 3xFLAG - HA - USP22 was immunoprecipitated using anti - HA - beads and the indicated co - immunoprecipitated proteins were analyzed by Western blotting . β - Actin served as a loading control .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A HeLa TRex RIPK3 CRISPR/Cas9 control (ctr) and USP22 KO cells were treated with 1 µg/ml Dox overnight. Protein expression of induced Strep-RIPK3 was analyzed by Western blotting. GAPDH served as loading control. The asterisk marks an unspecific band.B HeLa TRex RIPK3 control and USP22 KO cells were incubated with 1 µg/ml Dox for 18 h before pre-treatment with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα was added and cell death was measured after 4 and 5 h by analysis of PI-positive nuclei.C HeLa TRex RIPK3 control and USP22 KO cells were pre-treated with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 1, 2, 3, 4 and 5 h. Protein expression of phosphorylated RIPK1, total RIPK1, total RIPK3, phosphorylated MLKL, total MLKL and USP22, without (left) or with (right) 1 µg/ml Dox treatment overnight, was monitored by Western blotting. GAPDH was used as a loading control.D HT-29 control cells and RIPK3 KO cells re-expressing PAM-mutated Dox-inducible RIPK3 WT were incubated overnight with 1 µg/ml Dox. Cells were pre-treated with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 2 h, as indicated. Strep-RIPK3 was immunoprecipitated using anti‑Strep-beads and the indicated co-immunoprecipitated proteins were analyzed by Western blotting. β-Actin served as a loading control.E USP22 KO HT-29 cells and USP22 KO cells re-expressing PAM-mutated 3xFLAG-HA-USP22 were pre-treated with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα was added for 2 h, as indicated. 3xFLAG-HA-USP22 was immunoprecipitated using anti-HA-beads and the indicated co-immunoprecipitated proteins were analyzed by Western blotting. β-Actin served as a loading control.


Figure 4A HT - 29 control , USP22 KO and USP22 KO cells re - expressing PAM - mutated 3xFLAG - HA - USP22 WT or C185S were stimulated with 20 µM zVAD . fmk , 0 . 5 µM BV6 and 1 ng / ml TNFα for 4 h . Poly - ubiquitinated proteins were enriched by GST - TUBE pull - down , followed by incubation with the catalytic domain of USP2 , as indicated . RIPK3 and USP22 expression and levels of ubiquitinated RIPK3 were monitored using Western blotting with the indicated antibodies . β - Actin served as loading control . Ponceau - staining was used to confirm equal loading of GST - TUBE . B HT - 29 control and USP22 KO cells were transfected with His - ubiquitin for 24 h , as indicated . Cells were pre ‑ stimulated with 20 µM zVAD . fmk , 0 . 5 µM BV6 for 1 h . Following pre - treatment , 1 ng / ml TNFα was added for 4 h . His - ubiquitin was immunoprecipitated using Ni - NTA beads and detection of indicated proteins was performed by Western blotting . β - Actin served as loading control for the input , whereas His - ubiquitin levels served as loading control for immunoprecipitated ubiquitin . C HeLa TRex RIPK3 control and USP22 KO cells were incubated with 1 µg / ml Dox and transfected with HA - ubiquitin for 24 h , as indicated . Cells were pre - stimulated with 20 µM zVAD . fmk , 5 µM BV6 for 1 h . Following pre - treatment , 10 ng / ml TNFα were added for 3 h . HA - ubiquitin was immunoprecipitated using anti - HA - beads and detection of indicated proteins was performed by Western blotting . β - Actin served as loading control for the input , whereas HA - levels served as loading control for immunoprecipitated ubiquitin .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A HT-29 control, USP22 KO and USP22 KO cells re-expressing PAM-mutated 3xFLAG-HA-USP22 WT or C185S were stimulated with 20 µM zVAD.fmk, 0.5 µM BV6 and 1 ng/ml TNFα for 4 h. Poly-ubiquitinated proteins were enriched by GST-TUBE pull-down, followed by incubation with the catalytic domain of USP2, as indicated. RIPK3 and USP22 expression and levels of ubiquitinated RIPK3 were monitored using Western blotting with the indicated antibodies. β-Actin served as loading control. Ponceau-staining was used to confirm equal loading of GST-TUBE.B HT-29 control and USP22 KO cells were transfected with His-ubiquitin for 24 h, as indicated. Cells were pre‑stimulated with 20 µM zVAD.fmk, 0.5 µM BV6 for 1 h. Following pre-treatment, 1 ng/ml TNFα was added for 4 h. His-ubiquitin was immunoprecipitated using Ni-NTA beads and detection of indicated proteins was performed by Western blotting. β-Actin served as loading control for the input, whereas His-ubiquitin levels served as loading control for immunoprecipitated ubiquitin.C HeLa TRex RIPK3 control and USP22 KO cells were incubated with 1 µg/ml Dox and transfected with HA-ubiquitin for 24 h, as indicated. Cells were pre-stimulated with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. Following pre-treatment, 10 ng/ml TNFα were added for 3 h. HA-ubiquitin was immunoprecipitated using anti-HA-beads and detection of indicated proteins was performed by Western blotting. β-Actin served as loading control for the input, whereas HA-levels served as loading control for immunoprecipitated ubiquitin.


Figure 5B Bar graphs demonstrating the number of up ‐ , non ‐ , and downregulated ubiquitination sites in USP22 KO HT - 29 cells versus control under TBZ - treatment . D HeLa cells expressing Dox - inducible RIPK3 WT , D160N , K42R , K351 , K518R , 2xKR or 3xKR were pre ‑ incubated with 1 µg / ml Dox , 20 µM Dab , or 10 µM NSA overnight , followed by pre - treatment with 20 µM zVAD . fmk , 5 µM BV6 for 1 h . After pre - treatment , 10 ng / ml TNFα were added for 4 h . Cell death was measured by quantification of PI - positive nuclei . E HeLa cells expressing Dox - inducible RIPK3 mutants were treated with 1 µg / ml Dox overnight . Protein levels of inducible RIPK3 expression were analyzed by Western blotting . GAPDH served as loading control .

O B-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5B Bar graphs demonstrating the number of up‐, non‐, and downregulated ubiquitination sites in USP22 KO HT-29 cells versus control under TBZ-treatment.D HeLa cells expressing Dox-inducible RIPK3 WT, D160N, K42R, K351, K518R, 2xKR or 3xKR were pre‑incubated with 1 µg/ml Dox, 20 µM Dab, or 10 µM NSA overnight, followed by pre-treatment with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 4 h. Cell death was measured by quantification of PI-positive nuclei.E HeLa cells expressing Dox-inducible RIPK3 mutants were treated with 1 µg/ml Dox overnight. Protein levels of inducible RIPK3 expression were analyzed by Western blotting. GAPDH served as loading control.


Figure 6A HeLa cells expressing Dox - inducible RIPK3 WT , K518R or 3xKR were treated with 1 µg / ml Dox and / or 10 µM NSA and 20 µM Dab overnight . Protein levels of inducible RIPK3 expression and phosphorylated MLKL were examined by Western blotting . β - Actin was used as a loading control . B HeLa cells expressing Dox - inducible RIPK3 WT or 3xKR were treated with 1 µg / ml Dox overnight before pre - treatment with 20 µM zVAD . fmk , 5 µM BV6 for 1 h . After pre - treatment , 10 ng / ml TNFα were added for 1 , 2 , 3 and 4 h . Protein levels of inducible RIPK3 expression and phosphorylated MLKL were analyzed by Western blotting . β - Actin served as loading control . C HeLa cells expressing Dox - inducible RIPK3 WT , 2xKR or 3xKR were treated with 1 µg / ml Dox overnight before pre - treatment with 20 µM zVAD . fmk , 5 µM BV6 for 1 h . After pre - treatment , 10 ng / ml TNFα were added for 2 h . Protein levels of inducible RIPK3 expression were analyzed by Western blotting . β - Actin served as loading control . D HeLa cells expressing Dox - inducible RIPK3 WT , K518R or 3xKR were treated with 1 µg / ml Dox overnight before pre - treatment with 20 µM zVAD . fmk , 5 µM BV6 for 1 h . After pre - treatment , 10 ng / ml TNFα were added for 2 h . Whole cell lysates were generated using RIPA lysis buffer containing 2 % SDS . Protein levels of inducible RIPK3 expression were analyzed by Western blotting . β - Actin served as loading control . E HeLa cells expressing Dox - inducible RIPK3 WT , D160N , K518R or 3xKR were incubated overnight with 1 µg / ml Dox and pre - treated with 20 µM zVAD . fmk , 5 µM BV6 for 1 h . After pre - treatment , 10 ng / ml TNFα were added for 1 and 2 h . Strep - RIPK3 was immunoprecipitated by using anti - Strep - beads . Co - immunoprecipitated phosphorylated MLKL and RIPK1 , as well as protein expression of indicated proteins were analyzed by Western blotting . β - Actin served as a loading control . F HeLa cells expressing Dox - inducible RIPK3 WT , K518R or 3xKR were incubated with 1 µg / ml Dox and transfected with HA - ubiquitin for 24 h , as indicated . Cells were pre - treated with 20 µM zVAD . fmk , 5 µM BV6 for 1 h . After pre - treatment , 10 ng / ml TNFα were added for 2 h . HA - ubiquitin was immunoprecipitated using anti - HA - beads and detection of indicated proteins was performed by Western blotting . β - Actin served as loading control for the input , whereas HA - ubiquitin levels served as loading control for immunoprecipitated ubiquitin .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A HeLa cells expressing Dox-inducible RIPK3 WT, K518R or 3xKR were treated with 1 µg/ml Dox and/or 10 µM NSA and 20 µM Dab overnight. Protein levels of inducible RIPK3 expression and phosphorylated MLKL were examined by Western blotting. β-Actin was used as a loading control.B HeLa cells expressing Dox-inducible RIPK3 WT or 3xKR were treated with 1 µg/ml Dox overnight before pre-treatment with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 1, 2, 3 and 4 h. Protein levels of inducible RIPK3 expression and phosphorylated MLKL were analyzed by Western blotting. β-Actin served as loading control.C HeLa cells expressing Dox-inducible RIPK3 WT, 2xKR or 3xKR were treated with 1 µg/ml Dox overnight before pre-treatment with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 2 h. Protein levels of inducible RIPK3 expression were analyzed by Western blotting. β-Actin served as loading control. D HeLa cells expressing Dox-inducible RIPK3 WT, K518R or 3xKR were treated with 1 µg/ml Dox overnight before pre-treatment with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 2 h. Whole cell lysates were generated using RIPA lysis buffer containing 2% SDS. Protein levels of inducible RIPK3 expression were analyzed by Western blotting. β-Actin served as loading control.E HeLa cells expressing Dox-inducible RIPK3 WT, D160N, K518R or 3xKR were incubated overnight with 1 µg/ml Dox and pre-treated with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 1 and 2 h. Strep-RIPK3 was immunoprecipitated by using anti-Strep-beads. Co-immunoprecipitated phosphorylated MLKL and RIPK1, as well as protein expression of indicated proteins were analyzed by Western blotting. β-Actin served as a loading control.F HeLa cells expressing Dox-inducible RIPK3 WT, K518R or 3xKR were incubated with 1 µg/ml Dox and transfected with HA-ubiquitin for 24 h, as indicated. Cells were pre-treated with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 2 h. HA-ubiquitin was immunoprecipitated using anti-HA-beads and detection of indicated proteins was performed by Western blotting. β-Actin served as loading control for the input, whereas HA-ubiquitin levels served as loading control for immunoprecipitated ubiquitin.


Figure 7A HT ‑ 29 RIPK3 KO cells re - expressing Dox - inducible WT RIPK3 or the indicated RIPK3 mutants were incubated with 1 µg / ml Dox overnight . Cells were treated with 20 µM zVAD . fmk , 5 µM BV6 and 10 ng / ml TNFα for 4 h . Cell death was measured by analysis of PI - positive nuclei . B HT ‑ 29 RIPK3 KO cells re - expressing Dox - inducible WT RIPK3 or the indicated RIPK3 mutants were incubated with 1 µg / ml Dox overnight , as indicated . Cells were treated with 20 µM zVAD . fmk , 5 µM BV6 and 10 ng / ml TNFα for 2 h and analyzed by Western blotting for RIPK3 and phosphorylated MLKL expression levels . β - Actin served as a loading control .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A HT‑29 RIPK3 KO cells re-expressing Dox-inducible WT RIPK3 or the indicated RIPK3 mutants were incubated with 1 µg/ml Dox overnight. Cells were treated with 20 µM zVAD.fmk, 5 µM BV6 and 10 ng/ml TNFα for 4 h. Cell death was measured by analysis of PI-positive nuclei.B HT‑29 RIPK3 KO cells re-expressing Dox-inducible WT RIPK3 or the indicated RIPK3 mutants were incubated with 1 µg/ml Dox overnight, as indicated. Cells were treated with 20 µM zVAD.fmk, 5 µM BV6 and 10 ng/ml TNFα for 2 h and analyzed by Western blotting for RIPK3 and phosphorylated MLKL expression levels. β-Actin served as a loading control.


Figure 1A - D Proportional distribution of chymotrypsin - like activity of the proteasome across nine tissues from female ( n = 9 ) , and male ( n = 10 ) mice . Proportional distribution of trypsin - like activity of the proteasome across nine tissues from female ( n = 9 ) , and male ( n = 10 ) mice . Graphical representation of color code of tissues analyzed . Chymotrypsin - like activity of the proteasome across nine tissues in individual mice . Each data point ( n = 9 females and n = 10 males ) indicates data from a single mouse performed in triplicate . Each central line and error bar indicate mean + / - SEM . * indicates p < 0 . 05 by unpaired two - tailed students t - test comparing male and female samples . trypsin - like activity of the proteasome across nine tissues in individual mice . Each data point ( n = 9 females and n = 10 males ) indicates data from a single mouse performed in triplicate . Each central line and error bar indicate mean + / - SEM . * indicates p < 0 . 05 by unpaired two - tailed students t - test comparing male and female samples .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A-D Proportional distribution of chymotrypsin-like activity of the proteasome across nine tissues from female (n=9), and male (n=10) mice. Proportional distribution of trypsin-like activity of the proteasome across nine tissues from female (n=9), and male (n=10) mice. Graphical representation of color code of tissues analyzed.Chymotrypsin-like activity of the proteasome across nine tissues in individual mice. Each data point (n=9 females and n=10 males)indicates data from a single mouse performed in triplicate. Each central line and error bar indicate mean +/- SEM . * indicates p<0.05 by unpaired two-tailed students t-test comparing male and female samples.trypsin-like activity of the proteasome across nine tissues in individual mice. Each data point (n=9 females and n=10 males)indicates data from a single mouse performed in triplicate. Each central line and error bar indicate mean +/- SEM . * indicates p<0.05 by unpaired two-tailed students t-test comparing male and female samples.


Figure 2A - D Western blot analysis for K48Ub - linked proteins in spinal cord ( A ) and intestine ( C ) lysates from male and female mice . ( B , D ) Quantification of panels A and C , respectively . Values were normalized to actin as a loading control . Each central line and error bar indicate mean + / - SEM of a biological replicates . Data information : * indicates p < 0 . 05 by unpaired two - tailed students t - test comparing male and female samples . E ) Curve of correlation between chymotrypsin - like activity and Ub - K48 linked proteins in the intestine , where variability between mice is observed . F ) Native gel followed by western of β5 subunit to distinguish unassembled catalytic core ( CP ) or assembled with regulatory core ( RP ) in male ( n = 5 ) and females ( n = 5 ) mice in intestine . G ) Native gel followed by western blot of β5 subunit to distinguish unassembled catalytic core ( CP ) or assembled with regulatory core ( RP ) in male ( n = 5 ) and female ( n = 5 ) spinal cord . H ) Quantification of the ratio of assembled ( RP - CP ) versus unassembled ( CP ) proteasome in the intestine of males and females . Each central line and error bar indicate mean + / - SEM of a biological replicates ( n = 5 per group ) . I ) Quantification of the ratio of RP - CP versus CP proteasome in the spinal cord of males and females . Each central line and error bar indicate mean + / - SEM of a biological replicates ( n = 5 per group ) . Data information : * indicates p < 0 . 05 by unpaired two - tailed students t - test comparing male and female samples . J ) Correlation curve between chymotrypsin - like activity and RP - CP / CP ratio in individual female ( purple ) and male ( blue ) intestine . K ) Correlation curve between chymotrypsin - like activity and RP - CP / CP ratio in individual female ( purple ) and male ( blue ) spinal cord .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A-D Western blot analysis for K48Ub-linked proteins in spinal cord (A) and intestine (C) lysates from male and female mice. (B,D) Quantification of panels A and C, respectively. Values were normalized to actin as a loading control. Each central line and error bar indicate mean +/- SEM of a biological replicates. Data information: * indicates p<0.05 by unpaired two-tailed students t-test comparing male and female samples.E) Curve of correlation between chymotrypsin-like activity and Ub-K48 linked proteins in the intestine, where variability between mice is observed.F) Native gel followed by western of β5 subunit to distinguish unassembled catalytic core (CP) or assembled with regulatory core (RP) in male (n=5) and females (n=5) mice in intestine.G) Native gel followed by western blot of β5 subunit to distinguish unassembled catalytic core (CP) or assembled with regulatory core (RP) in male (n=5) and female (n=5) spinal cord.H) Quantification of the ratio of assembled (RP-CP) versus unassembled (CP) proteasome in the intestine of males and females. Each central line and error bar indicate mean +/- SEM of a biological replicates (n=5 per group). I) Quantification of the ratio of RP-CP versus CP proteasome in the spinal cord of males and females. Each central line and error bar indicate mean +/- SEM of a biological replicates (n=5 per group). Data information: * indicates p<0.05 by unpaired two-tailed students t-test comparing male and female samples.J) Correlation curve between chymotrypsin-like activity and RP-CP/CP ratio in individual female (purple) and male (blue) intestine. K) Correlation curve between chymotrypsin-like activity and RP-CP/CP ratio in individual female (purple) and male (blue) spinal cord.


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

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


Figure 4B ) Western blot analysis of Ub - K48 proteins in the soluble fractions of intestine lysates from mice plus or minus heat shock . C ) Quantification of panel B . Each central line and error bar indicate mean + / - SEM of a biological replicates ( n = 3 biological replicates per group ) . Data information : * indicates p < 0 . 05 , * * indicates p < 0 . 005 , and * * * indicates p < 0 . 0005 by unpaired two tailed students t - test comparing male and female samples or samples with or without heat shock . D ) Western blot analysis of Ub - K48 proteins in the insoluble fraction of intestine lysates from mice plus or minus heat shock . E ) Quantification of D . Each central line and error bar indicate mean + / - SEM of a biological replicates ( n = 3 biological replicates per group ) Data information : * indicates p < 0 . 05 , * * indicates p < 0 . 005 , and * * * indicates p < 0 . 0005 by unpaired two tailed students t - test comparing male and female samples or samples with or without heat shock . F ) Western blot analysis of Ub - K48 proteins in the soluble fractions of spinal cord lysates from mice plus or minus heat shock . G ) Quantification of F . Each central line and error bar indicate mean + / - SEM of a biological replicates ( n = 3 biological replicates per group ) Data information : * indicates p < 0 . 05 , * * indicates p < 0 . 005 , and * * * indicates p < 0 . 0005 by unpaired two tailed students t - test comparing male and female samples or samples with or without heat shock . H ) Western blot analysis of Ub - K48 proteins in the insoluble fraction of spinal cord lysates from mice plus or minus heat shock . I ) Quantification of H . Each central line and error bar indicate mean + / - SEM of a biological replicates ( n = 3 biological replicates per group ) Data information : * indicates p < 0 . 05 , * * indicates p < 0 . 005 , and * * * indicates p < 0 . 0005 by unpaired two tailed students t - test comparing male and female samples or samples with or without heat shock . K ) Western blot of Ub - K48 proteins in the insoluble fraction of protein lysates of SOD1 - G93A mouse spinal cord plus or minus heat shock . L ) Quantification of panel K . Each central line and error bar indicate mean + / - SEM of a biological replicates ( n = 5 biological replicates per group ) Data information : * indicates p < 0 . 05 , * * indicates p < 0 . 005 , and * * * indicates p < 0 . 0005 by unpaired two tailed students t - test comparing male and female samples or samples with or without heat shock . M ) Chymotrypsin - like proteasome activity in spinal cord lysates from SOD1 - G93A mice . Each central line and error bar indicate mean + / - SEM of a biological replicates ( n = 5 biological replicates per group ) . Bordered points indicate biological replicates and un - bordered points indicate technical replicates . Data information : * indicates p < 0 . 05 , * * indicates p < 0 . 005 , and * * * indicates p < 0 . 0005 by unpaired two tailed students t - test comparing male and female samples or samples with or without heat shock . N ) Filter trap assay of protein lysates from the insoluble fraction of SOD1 - G93A spinal cord lysates plus or minus heat shock . O ) Quantification of panel N . Each central line and error bar indicate mean + / - SEM of a biological replicates ( n = 5 biological replicates per group ) Data information : * indicates p < 0 . 05 , * * indicates p < 0 . 005 , and * * * indicates p < 0 . 0005 by unpaired two tailed students t - test comparing male and female samples or samples with or without heat shock . P ) Representative transmission electron micrographs of protein aggregates in the insoluble fraction of SOD1 - G93A spinal cord lysates plus or minus heat shock . Scale bar equals 600nm . Q ) Quantification of P . ( n = 9 technical replicates per group ) Data information : * indicates p < 0 . 05 , * * indicates p < 0 . 005 , and * * * indicates p < 0 . 0005 by unpaired two tailed students t - test comparing male and female samples or samples with or without heat shock .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4B) Western blot analysis of Ub-K48 proteins in the soluble fractions of intestine lysates from mice plus or minus heat shock. C) Quantification of panel B. Each central line and error bar indicate mean +/- SEM of a biological replicates (n=3 biological replicates per group). Data information: * indicates p<0.05, ** indicates p<0.005, and *** indicates p<0.0005 by unpaired two tailed students t-test comparing male and female samples or samples with or without heat shock.D) Western blot analysis of Ub-K48 proteins in the insoluble fraction of intestine lysates from mice plus or minus heat shock. E) Quantification of D. Each central line and error bar indicate mean +/- SEM of a biological replicates (n=3 biological replicates per group) Data information: * indicates p<0.05, ** indicates p<0.005, and *** indicates p<0.0005 by unpaired two tailed students t-test comparing male and female samples or samples with or without heat shock.F) Western blot analysis of Ub-K48 proteins in the soluble fractions of spinal cord lysates from mice plus or minus heat shock. G) Quantification of F. Each central line and error bar indicate mean +/- SEM of a biological replicates (n=3 biological replicates per group) Data information: * indicates p<0.05, ** indicates p<0.005, and *** indicates p<0.0005 by unpaired two tailed students t-test comparing male and female samples or samples with or without heat shock.H) Western blot analysis of Ub-K48 proteins in the insoluble fraction of spinal cord lysates from mice plus or minus heat shock. I) Quantification of H. Each central line and error bar indicate mean +/- SEM of a biological replicates (n=3 biological replicates per group) Data information: * indicates p<0.05, ** indicates p<0.005, and *** indicates p<0.0005 by unpaired two tailed students t-test comparing male and female samples or samples with or without heat shock.K) Western blot of Ub-K48 proteins in the insoluble fraction of protein lysates of SOD1-G93A mouse spinal cord plus or minus heat shock. L) Quantification of panel K. Each central line and error bar indicate mean +/- SEM of a biological replicates (n=5 biological replicates per group) Data information: * indicates p<0.05, ** indicates p<0.005, and *** indicates p<0.0005 by unpaired two tailed students t-test comparing male and female samples or samples with or without heat shock.M) Chymotrypsin-like proteasome activity in spinal cord lysates from SOD1-G93A mice. Each central line and error bar indicate mean +/- SEM of a biological replicates (n=5 biological replicates per group). Bordered points indicate biological replicates and un-bordered points indicate technical replicates. Data information: * indicates p<0.05, ** indicates p<0.005, and *** indicates p<0.0005 by unpaired two tailed students t-test comparing male and female samples or samples with or without heat shock.N) Filter trap assay of protein lysates from the insoluble fraction of SOD1-G93A spinal cord lysates plus or minus heat shock. O) Quantification of panel N. Each central line and error bar indicate mean +/- SEM of a biological replicates (n=5 biological replicates per group) Data information: * indicates p<0.05, ** indicates p<0.005, and *** indicates p<0.0005 by unpaired two tailed students t-test comparing male and female samples or samples with or without heat shock.P) Representative transmission electron micrographs of protein aggregates in the insoluble fraction of SOD1-G93A spinal cord lysates plus or minus heat shock. Scale bar equals 600nm. Q) Quantification of P. (n=9 technical replicates per group) Data information: * indicates p<0.05, ** indicates p<0.005, and *** indicates p<0.0005 by unpaired two tailed students t-test comparing male and female samples or samples with or without heat shock.


Figure 1A . 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 . ( 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 . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A. 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.(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.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.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.


Figure 2A . 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 ) . 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 ) . 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 . 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 . 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 . 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 . 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 ) . 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 B-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. 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). 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).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.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.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. 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.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).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.


Figure 3Plasma ( 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 . 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 ) . ( 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 . 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 . Blood glucose following the administration of HFD ( wt + HFD , n = 10 ; LowOXPHOS + HFD , n = 10 ) . T2D onset occurred on day 80 in wt and day 60 in LowOXPHOS mice . P . Insulin ( ITT ) and glucose ( GTT ) tolerance tests on day 60 of HFD ( wt + HFD , n = 10 ; LowOXPHOS + HFD , n = 10 ) . LowOXPHOS but not wt mice appeared diabetic .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Plasma (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.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).(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.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. 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.


Figure 4A . 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 . 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 ) . 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 . 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 . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A. 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.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).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.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.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.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.


Figure 5A . 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 . ( 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 . 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 . 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A. 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.(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.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.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.


Figure 6 ( 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(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.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.


Figure 7A . 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 . 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 ) . 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 . 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 . 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 . K . GTT after 80 days of HFD ( left ) and ITT after 90 days of HFD ( right ) in wt and LowOXPHOS mice treated with edaravone , showing that the compound has an improving effect itself . Data information : H - L : wt + HFD , n = 5 ; LowOXPHOS + HFD n = 4 ; wt + HFD + edaravone , n = 10 ; LowOXPHOS + HFD + edaravone n = 9 . Bars are the mean ± s . e . m . of the indicated ( n ) mice / genotype . * p < 0 . 05 when compared to wt by ANOVA and Student ' s t - test . L . Blood glucose following the administration of HFD . T2D onset occurred on day 90 in wt and LowOXPHOS mice treated with edaravone . Data information : H - L : wt + HFD , n = 5 ; LowOXPHOS + HFD n = 4 ; wt + HFD + edaravone , n = 10 ; LowOXPHOS + HFD + edaravone n = 9 . Bars are the mean ± s . e . m . of the indicated ( n ) mice / genotype . * p < 0 . 05 when compared to wt by ANOVA and Student ' s t - test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A. 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.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).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.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.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.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.