Fig. 2
Fam3a is a direct Stat3 and MyoD target. a Venn diagram of the overlap among the differentially expressed genes between the different comparisons in the RNA-seq analysis. b Quantitative real-time PCR analysis of the top 10 selected candidates in the RNA-seq samples (n = 3 animals). The red line indicates the average expression levels in control samples. c Fam3a mRNA levels in freshly isolated activated muscle stem cells (MuSCs) (3 dpi) from Pax7-CreER;Stat3f/f mice and control littermates (different samples from RNA-seq) (n = 3–4 animals). d Gene expression analysis of MuSCs cultured in growth media (GM) or differentiation media (DM) for 72 h (n = 3 independent experiments). e Fam3a promoter analysis using JASPAR identified one putative Stat3-binding site. Chromatin immunoprecipitation (ChIP) assay in C2C12 myoblasts incubated in the presence or absence of interleukin-6 (100 ng/ml) to monitor the recruitment of Stat3 and the presence of histone H3 acetylated at lysine 27 (H3K27Ac) in the Fam3a promoter (n = 4 independent experiments). f MyoD ChIP-seq signal and peak distribution on the Fam3a promoter on C2C12 myotubes. Previously published data were used for the analysis40. g MyoD ChIP-seq signal distribution and peaks on the FAM3A promoter on IMR90-derived myoblasts and myotubes. h Fam3a mRNA levels in wild type and MyoD−/− MuSCs cultured in growth conditions for 72 h (n = 4 independent experiments). i Reporter assay in HEK293 cells transfected with plasmids encoding Stat3, MyoD, and/or an empty vector as control, together with the reporter vector containing the luciferase reporter gene under the control of the Fam3a promoter (n = 12 independent experiments). Data are represented as mean ± SEM (Student’s t test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)
