Figure 6

Thalidomide (THL) inhibits TGF-β1-mediated phosphorylation of Smad2 and Smad3 in CCL-149 cells. (A) CCL-149 cells were transfected with Smad2 and Smad3 siRNAs. Smad2 and Smad3 siRNA treatment resulted in approximately a 60% decrease in the density of Smad protein bands 48 hours after transfection. (B) Cells transfected with Smad2 siRNA and/or Smad3 siRNA were treated with or without TGF-β1 (5 ng/mL) for 48 h. Cells were lysed and α-SMA levels were evaluated by western blot. (C) Cells were stimulated with TGF-β1 (5 ng/mL) in the presence of vehicle, SB431542 (5 μM) or increasing doses of THL (1, 10, and 100 μg/mL) for 48 h. Then, cells were lysed and the phosphorylation of Smad2 and Smad3 was evaluated by western blot. β-Actin was used as a loading control. Data are expressed as the means ± SD, and n = 3 in each group. THL: thalidomide; SB431542: TGF-β1R-Kinase inhibitor; α-SMA: α-smooth muscle actin; ^#p < 0.05, ^##p < 0.01 compared to the TGF-β1 group (untreated); ^*p < 0.05, ^**p < 0.01 compared to all other groups.