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.