Figure 3. Smad signaling mediates senescence induction of a myofibroblastic phenotype. Western blot showing SMA and phospho(p)-Smad2/3 expression following treatment of HFFF2 fibroblasts with TGF-β1 (A) or irradiation (B) (Smad2 and HSC-70 as loading controls). (C) Representative images of immunofluorescence on HFFF2 fibroblasts showing expression of SMA (green), pSmad2/3 (red), SA-β-Galactosidase activity (grey; in bright field) with DAPI nuclear counterstain (Blue) (scale bar indicates 100µm). (D) Western blots showing SMAD3 knock-down (Tot-FAK as loading control) (left) and SMA expression (HSC-70 as loading control; right) in irradiated HFFF2 cells. (E) Western blot for SMA expression in irradiated HFFF2 pre-treated with a pan TGF-β1 inhibitory antibody. (F) TGFβ-1 assay showing luciferase activity controlled by a TGFβ-1 responsive promoter in MLEC cells co-cultured for 24 hours with untreated or irradiated fibroblasts (y axis indicates the ratio between luciferase activity and HFFF2s protein concentration). Data are presented as mean ± SEM and statistics are shown for T-test compared to controls. See also Supplementary Fig. S3.