Sc 514544x

Total protein was extracted with cell lysis buffer (Beyotime) supplemented with protease and phosphatase inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany), and the concentration was measured with BCA kit (Beyotime). The proteins were separated with 12% SDS-PAGE and transferred to PVDF membranes (ThermoScientific). The membranes were separately incubated with primary antibodies overnight at 4 °C, and anti-rabbit (A7016) or anti-mouse (A0216) HRP-conjugated secondary antibodies (Beyotime) for 1 h at room temperature^{49 (link)}. Subsequently, the membranes were washed and the signals were visualized with Clarity ECL Substrate (Bio-Rad, Hercules, CA, USA). Gray-scale results were normalized against β-actin for semiquantitative analysis. The antibodies against IRF1 (sc-514544x), PPARα (sc-130640), and β-actin (sc-47778) were bought from Santa Cruz Biotechnology (Dallas, TX, USA). The antibodies against PGC1α (ab54481), Pit1 (ab10545), Pit2 (ab191182), phosphorylated FGFR1 (p-FGFR1, ab59194), total FGFR1 (t-FGFR1, ab824), phosphorylated FGFR4 (p-FGFR4, ab192589), total FGFR4 (t-FGFR4, ab41948), NRF1 (ab175932), and TFAM (ab131607) were obtained from Abcam Biotechnology (Cambridge, MA, USA). All of the antibodies were diluted 1:1000 for western blot. Uncropped and unprocessed scans of all blots are provided in Supplementary Figs. 17–21 in the Supplementary Information.

Total protein was extracted using a cell lysis buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS (Beyotime, Shanghai, China) supplemented with each protease and phosphatase inhibitor cocktail tablet (Roche Diagnostics GmbH, Mannheim, Germany) per 10 ml solution. Western blot was performed as previously described 57 (link). Primary antibodies against IRF1 (sc-514544x) and β-actin (sc-47778) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The antibodies against P62 (5114), p-mTOR (5536), t-mTOR (2983), p-p70S6K (9234), p-4EBP1 (2855), PINK1 (6946) and Parkin (4211) were obtained from Cell Signaling Technology (CST, Danvers, MA, USA). The antibodies against DRP1 (ab56788), MFN1 (ab57602), MFN2 (ab50838), OPA1 (ab90857), FIS1 (ab71498), AhR (ab2769), COXIV (ab14744), PGC1α (ab54481) and TFAM (ab131607) were purchased from Abcam (Cambridge, MA, USA). LC3 antibody (L7543) was purchased from Sigma-Aldrich.

H9c2 cells were treated with control or HP for 24 h, and then fixed with 1% formaldehyde for 10 min. The cells were lyzed in SDS lysis buffer. The chromatin was sonicated to shear DNA to an average length between 200 to 1000 bp, and immunoprecipitated with 2 μg antibody against IRF1 (sc-514544x, Santa Cruz Biotechnology), taking IgG as a negative control. The precipitated DNA was amplified by PCR and qPCR with the primers (−709 to −511) that cover the IRF1 binding sites (−632 to −612). Primers (−1711 to −1598) without IRF1 binding sites served as a negative control, while the total DNA (Input) served as a positive control. For acetylation detection, the sonicated DNA was immunoprecipitated with 2 μg antibody against H3 (Lys9) (#9671) or H4 (Lys12) (#13944) (Cell Signaling Technology, Danvers, MA, USA), taking IgG as a negative control. The primers for ChIP are listed in Supplementary Table 9.