Figure 6. STAT3 regulated FOXO1 expression is responsible for C26 CM-induced myotube atrophy.
(a) Western blot analysis of FOXO1 expression in skeletal muscle from C26 cachectic mice and C2C12 myotubes with or without 17DMAG treatment. (b) Representative Western blot assay of the FOXO1 expression after knocking down STAT3 by siRNA. (c)Representative immunofluorescence microscopy images showing the subcellular localization of FOXO1 in C2C12. (d) Schematic structure of the FoxO1 promoter region (NCBI Reference Sequence: NC_000069.5 for FoxO1). The transcription start site (TSS) is denoted by a black arrow. (e-f) Chromatin immunoprecipitation (CHIP) analysis was performed with C2C12, and STAT3 binding to the FoxO1 promoter region was analyzed. Cell lysates were immunoprecipitated with anti-STAT3 Ab or control IgG. Immunoprecipitated and input DNA was analyzed by semi-quantitative PCR (e) or qPCR (f)using primers corresponding to FoxO1 promoter sites. (g)Representative Western blot assay of FOXO1, p-FOXO1, and atrophy genes expression in C2C12 myotube co-transfected with FOXO1 siRNA and STAT3-C recombinant plasmid. (h) Representative Western blot of FOXO1, p-FOXO1, and atrophy genes expression in C2C12 myotube transfected with or without mutated forms (constitutive activated) of STAT3 overexpression plasmid (Stat3-C) and treated with or without 17DMAG.