Fig. 5: Smad3 signaling regulates Srsf1 expression and EDB(+) FN levels.

a Representation of transcription factor binding sites up to 1 kb upstream of the mouse Srsf1 start codon. The p-value shows the probability of obtaining the same interaction score on a random sequence. b Multiple sequence alignment of the Smad3 binding site in the Srsf1 promoter from 9 different vertebrate species. Letter size indicates conservation and asterisks show identical nucleic acids. c Schematic of the mouse Srsf1 promoter around the Smad3 (yellow box, red fonts) and Smad5 (gray box, red fonts) binding motif and chromatin immunoprecipitation (ChIP) using MuSC-derived myoblasts with p-Smad3 and p-Smad1/5/9 antibodies compared to the isoform IgG and no antibody (noAB) controls. ChIP primer pairs in the schematic are indicated by arrows. TSS = Transcription start site. Readouts were normalized to input. d–f Representative immunostaining and quantification of nuclear fluorescence intensity of p-Smad3 (green) and Srsf1 (red) in freshly isolated cultured MuSCs and early passage FAPs from young mice. DNA (blue) was counterstained using DAPI. Scale bar = 10 µm. g Schematic showing the molecular structure and mechanism of action of the cell-permeable inhibitor of TGFβ1-dependent Smad3 phosphorylation SIS3. h,i Quantification of the effect of increasing concentrations of SIS3 on Srsf1 and EDB(+) FN expression in MuSC-derived myoblasts by semi-quantitative PCR. j–m Representative images and quantification of Pax7+ (green), and Ki67+ (red) MuSC-derived myoblasts treated with increasing concentrations of SIS3. DNA (blue) was counterstained using DAPI. Scale bar = 200 µm. Bars represent means ± SEM from myoblasts isolated from n = 3 biological replicates per condition, each from separate young mice. P-values were calculated using a two-tailed Z-test (a), a one-tailed Student’s t-test (e, f), or one-way ANOVA with Dunnett’s (c, h, I, k–m) post-hoc test. ****p < 0.0001, ***p < 0.001. **p < 0.01. *p < 0.05. Source data are provided as a Source Data file.