Fig. 3: TGF-β1-induced suppression of Mast4 enhances chondrogenesis by increasing Sox9-Smad3 association.

a Sox9 and Smad3 were co-transfected to wild-type and Mast4-depleted C3H10T1/2 cells, followed by TGF-β1 treatment (5 ng/ml for 30 min). Sox9 was immunoprecipitated using HA antibody. Band intensities representing GFP-Smad3 and HA-Sox9 expression level in the immunoprecipitates were converted by densitometry using ImageJ software into the ratio of Smad3 to Sox9. Data are reported as mean ± SD of three independent experiments (n = 3). Unpaired two-tailed Student’s t test (P < 0.05) was conducted for statistical analysis. b Smad3 ChIP on Col2a1 gene assay was performed in differentiating C3H10T1/2 cells for 6 days. c Mast4 expression was examined by western blot and RT-PCR of C3H10T1/2 cells treated with TGF-β1 for 48 h. d Luciferase assay was conducted in undifferentiated C3H10T1/2 cells. Vactosertib (0.5 μM) was pre-treated for 2 h prior to TGF-β1 (5 ng/ml) treatment for 24 h. e Smad3 ChIP assay on Mast4 gene was conducted in C3H10T1/2 cells undergoing chondrogenic differentiation for 6 days. TGF-β1 (5 ng/ml) and Vactosertib (0.5 μM) were treated for 48 h and 50 h, respectively, before harvest. f Endogenous Mast4 and Sox9 protein expression was examined by western blotting in differentiating C3H10T1/2 cells. g TGF-β1 (5 ng/ml) was treated for 24 h and 48 h, and Vactosertib (0.5 μM) was treated for 48 h to human primary chondrocytes. b, d and e Data are representative mean ± SD of three independent experiments, each conducted in triplicate (n = 3). Unpaired two-tailed Student’s t test (P < 0.05) with Benjamini-Hochberg correction for multiple tests was conducted for all statistical analyses. a, c, f and g The representative results were obtained from at least three independent experiments. TCL: total cell lysates. Uncropped blots in a, c, f and g are shown in Source Data file. Source data for a, b, d and e are provided as a Source Data file.