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).