Figure 3

ATO inhibits Smad2/Smad3 and Akt phosphorylation but does not affect p38 phosphorylation. (A-B) NHLFs were pre-treated with ATO for 24 hrs. Then cells were treated with TGF-β1 (1 ng/ml) for 30 mins. TGF-β1 did not affect total Smad2 and Smad3 expression, but did increase their phosphorylation. TGF-β1 induced Smad2 and Smad3 phosphorylation were blocked by ATO. (C) NHLFs were pre-treated with ATO for 24 hrs, and then washed 5 times with PBS to remove ATO from the media. Thereafter cells were exposed to TGF-β1 (1 ng/ml) for 30 mins. The results indicate that the effects of ATO on TGF-β1 induced Smad2 and Smad3 phosphorylation are not due to disruption of the of the TGF-β1 ligand. (D) NHLFs were pre-treated with ATO for 24 hrs., and then treated with TGF-β1 (1 ng/ml) for 12 hrs. Akt phosphorylation was induced by TGF-β1 and this induction was diminished by ATO. (E) NHLFs were pre-treated with ATO for 24 hrs, then treated with TGF-β1 (1 ng/ml) for 30 mins. ATO increased TGF-β1 induced p38 phosphorylation. (F) TGF-β1 induces H₂O₂ in NHLFs. ATO at the indicated concentrations did not induce H₂O₂, but did diminish TGF-β1-induced H₂O₂ production. (G) NHLFs were pre-treated with ATO for 24 hrs, then treated with TGF-β1 (1 ng/ml) with or without H₂O₂ for 30 mins. Smad3 phosphorylation was up-regulated by H₂O_2.(H) TGF-β1 induced NOX-4 mRNA expression in NHLFs, whereas ATO at the indicated concentrations inhibited TGF-β1-induced NOX-4 mRNA expression. Western blot data represents consistent trend in three independent repeats with the best image quality. RT-PCR data represent results from three independent experiments with duplicate repeats. *P value < 0.05, **P value <0.01, ***P value < 0.001.