Fig. 2. Antioxidants downregulate TGFß1-mediated expression of SMA. (A) Subconfluent HDFs were cultured in CM^HDF and either untreated or pretreated for 4 hours with 5.0 mM NAC or for 24 hours with 0.5 µM Na₂SeO₃ before addition of 10 ng/ml rTGFß1. TGFß1 and the antioxidants were present for an additional 48 hours. The level of SMA protein was determined by western blot. Three independent experiments were performed. (B) HDF monolayer cultures were cultured in CM^HDF either containing 0.6 nM SeP for 48 hours before treatment with 10 ng/ml TGFß1 for a further 48 hours or containing SeP for the complete time of 96 hours. The level of SMA protein was determined by western blot. The densitometric analysis describes protein expression as a percentage, setting rTGFß1-treated controls at 100%. The experiments were performed in duplicate. (C,D) Subconfluent HDFs were cultured in CM^HDF and either untreated or pretreated for 24 hours with 0.5 µM Na₂SeO₃ (C) or ebselen (D) before addition of 10 ng/ml rTGFß1. TGFß1 and selenite or ebselen were present for an additional 48 hours. The levels of cytosolic glutathione peroxidase (GPx) (C) and of SMA (D) were detected by western blot. -tubulin was used as loading control. Quantitative data were standardized to -tubulin and densitometric values represent fold increase over control, which was set at 1.0. Data are representative of two independent experiments.