Nrf2 inhibits the TGF-β/Smad3 signaling pathway.

(A and B) Representative Western blot analysis of the effect of Ad-Nrf2 on Smad3 phosphorylation in TGF-β-stimulated NRK-49F (A) and RMC (B) cells. Cells were serum-starved for 24 h after infection with Ad-GFP or Ad-Nrf2 and then stimulated with TGF-β for 1 h before harvesting for Western blot analysis. Quantitative analysis of p-Smad3 to total Smad3 ratio. Data in bar graph are the mean ± SE of three independent measurements. (A) ^*P<0.01 vs. control, ^**P<0.05 vs. TGF-β stimulation. (B) ^*P<0.001 vs. control, ^**P<0.05, ^#P<0.01 vs. TGF-β stimulation. (C) Representative semi-quantitative RT-PCR analysis of the effect of Nrf2-siRNA on DMF-induced Nrf2 mRNA expression. AD-293 cells were transfected with a control scrambled siRNA (Control-siRNA) or Nrf2-siRNA and incubated for 24 h. Cells were incubated in serum-free media for 12 h, treated with DMF (40 µmol/l) for 1 h, and harvested for semi-quantitative RT-PCR. (D) The effect of Nrf2-siRNA on DMF-induced suppression of TGF-β-stimulated (CAGA)₉MLP-Luc activity. AD-293 cells were serum-starved for 12 h after cotransfection with (CAGA)₉MLP-Luc and the Control-siRNA or Nrf2-siRNA for 24 h. Cells were then stimulated with TGF-β for 5 h after pretreatment with DMF (40 µmol/l) for 1 h. Data represent the means ±SEM of three independent measurements. *P<0.01 vs. reporter alone, ^**P<0.05 vs. TGF-β stimulation, and ^***P<0.05 vs. Control-siRNA with DMF treatment. (E) Representative semi-quantitative RT-PCR analysis of the effect of Nrf2-siRNA on DMF-induced inhibition on TGF-β-stimulated type I collagen mRNA expression. NRK-49F cells were transfected with Control-siRNA or Nrf2-siRNA for 24 h, and then starved for 12 h. Cells were pretreated with DMF (10 µmol/l) for 1 h, stimulated with TGF-β for 24 h, and then harvested for semi-quantitative RT-PCR analysis.