Fig. 4. FOXO1 expression via TGFβ1 is mediated by SMAD3 and repressed by SMAD2.

Chondrocytes were incubated with or without TGFβ1 (10 ng/ml) for 24 hours after transfection with siCtrl or siSMAD3. Relative SMAD3 (A) and FOXO1 (B) mRNA levels were measured by qRT-PCR. Gene expression at each stage is expressed relative to the level in cells transfected with siCtrl and incubated without TGFβ1; n = 8 independent experiments. (C) Levels of total FOXO1 and SMAD3 were analyzed by western blotting. GAPDH was used as a loading control. Graphs show total levels of FOXO1, relative to the corresponding levels in cells transfected with siCtrl and incubated without TGFβ1; n = 8 independent experiments. (D) SMAD3 and FOXO1 proteins were visualized by immunocytochemistry. SMAD3 is shown in green, and FOXO1 is shown in red. Nuclei were detected with DAPI (blue staining). Bars, 50 μm. Chondrocytes were incubated with or without TGFβ1 (10 ng/ml) for 24 hours after transfection with siCtrl or siSMAD2. Relative SMAD2 (E) and FOXO1 (F) mRNA levels were measured by qRT-PCR. Gene expression at each stage is expressed relative to the level in cells transfected with siCtrl and incubated without TGFβ1; n = 9 independent experiments. (G) Levels of total FOXO1 and SMAD2 were analyzed by western blotting. Graphs show total levels of FOXO1, relative to the corresponding levels in cells transfected with siCtrl and incubated without TGFβ1; n = 8 independent experiments. (H) SMAD2 and FOXO1 proteins were visualized by immunocytochemistry. SMAD2 is shown in green, and FOXO1 is shown in red. Bars, 50 μm. Data are presented as dots showing individual values and as means ± S.D. Statistical analysis was performed using one-way repeated measures ANOVA with the Tukey–Kramer post hoc test. *P < 0.05.