Chromatin immunoprecipitation (ChIP) analysis
The binding site of STAT3 to the promoter region of FOXO1 is predicted by CiiiDER software according to the JASPAR database (Fornes et al., 2020; Gearing et al., 2019). The ChIP assay was performed according to the manufacturer’s instructions (P2078,Beyotime,China). C2C12 cells were fixed with 1% formaldehyde at 37°C for 10 min, lysed, and sonicated. Soluble chromatin was coimmunoprecipitated with anti-STAT3 antibodies or an equal amount of rabbit IgG. The immunoprecipitated and input DNA was subjected to PCR and qPCR using FOXO1 promoter site-specific primers (5’-CGACTTCAACACCTCATCGCTTC-3’ and 5’-AGGCGCGCAGATCCTTCGGTGA-3’). Q-PCR was performed with Power SYBR Green PCR Master Mix (Life Technologies, New York, NY). The fold change in enrichment relative to the input DNA was calculated using the comparative Ct method (∆∆Ct).