Fig. 4. Activin directly regulates pro-fibrotic ECM gene expression in fibroblasts.

a qRT-PCR (relative to Rps29) using RNA from primary murine neonatal fibroblasts treated with activin A (20 ng/ml) or vehicle for 1.5 and 3 h. n = 3. b qRT-PCR using RNA from murine fibroblasts from 5-day wounds of adult WT and Act mice treated with activin A (20 ng/ml) or vehicle for 3 h. n = 8, 9, 6, 9 for WT-control, WT-Activin, Act-Control, and Act-Activin, respectively. c Chromatin immunoprecipitation from lysates of immortalized mouse fibroblasts treated with activin A (20 ng/ml) or vehicle for 6 h. Antibodies against anti-histone H3 (H3) or Smad2/3 (Smad) or normal rabbit IgG (IgG) were used. The bound DNA was amplified using primers spanning conserved Smad-binding elements (SBEs) in the promoter regions of Postn and Aspn (see Supplementary Fig. 4b). Representative agarose gels are shown, with the size marker and respective inputs. The experiment was repeated twice with similar results (see also Supplementary Fig. 4c). d Representative photomicrographs of human primary foreskin fibroblasts treated with activin A (20 ng/ml) or vehicle for 24 h and stained for collagen type I or fibronectin (red) (nuclei were counterstained with Hoechst (blue)). Bar graphs show quantification of the percentage of the stained area. Scale bars: 200 μm. n = 3. Graphs show mean ± SEM and P values; mean expression levels in control cells (a) or control cells in WT mice (b) were set to 1; two-tailed Student's t-test (a, d), one-way ANOVA and Tukey’s multiple comparison post hoc tests (b). All n numbers indicate biological replicates. Source data are provided as a Source Data file (Fig. 4).