Pgc 1α

APAP (Bioxtra, ≥99.0%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). RPMI-1640 medium, dual antibodies (penicillin and streptomycin), PBS, and 0.25% trypsin were purchased from HyClone. Fetal bovine serum was purchased from Gibco. CCK-8 was purchased from Nanjing Enjing Biotechnology. An Annexin V/PI double staining apoptosis kit and nuclear protein extraction kit were purchased from Nanjing Kaiji Biotechnology. Autophagy double standard adenovirus reagent was purchased from Han Heng Biotechnology. A BCA protein quantitative kit, JC-1 and MitoSox were purchased from Thermo Fisher. MitoTracker Red and transfection reagent were purchased from Invitrogen. The qPCR kit was purchased from AG Biotechnology, and the small interfering RNA product was purchased from Beijing Qing Ke Biotechnology. The primary antibodies used in this study included PARK7 (Abcam, #ab76008), LC3B (Abcam, #ab192890), P62 (Abcam, #ab109012), Keap1 (Abcam, #ab227828), Nrf2 (Abcam, #ab62352), PGC1-α (Affinity, #AF5395), NRF1 (Affinity, #AF5298), TFAM (Affinity, #AF0531), β-tubulin, actin, and TBP antibodies and goat anti-rabbit IgG and goat anti-mouse IgG antibodies, which were purchased from Affinity. All other chemicals were analytical grade chemicals provided by commercial suppliers.

Western blotting was performed as previously reported [22 ]. LD muscle samples were homogenised in RIPA lysis buffer (Beyotime biotechnology, Shanghai, China) containing a protease inhibitor (Roche, Shanghai, China). Proteins were separated on 10% SDS–PAGE gel and then were transferred onto a PVDF membrane (Bio-Rad, Shanghai, China). The membrane was blocked with 5% skimmed milk for 1 h at room temperature, and then incubated with the respective primary antibody overnight at 4 °C. Anti-slow MyHC (Sigma, Cat. No. M8421), anti-fast MyHC (Sigma, Cat. No. M4276), PGC-1α (Affinity Biosciences, Cat. No. AF5395) and GAPDH (Absin, Cat. No. abs132004) were used. The membranes were washed six times, and subsequently incubated with secondary antibodies (CST) (1:2000 dilution in 5% milk/1 × TBST) for 1 h. Proteins were detected using an ECL reagent (Bio-Rad, Shanghai, China) on a Molecular Imager ChemiDoc XRS + System (Bio-Rad). The western blots were quantified using the ImageJ software (National Institutes of Health).