FIGURE 2.

Mitochondrially targeted antioxidants inhibit TGF-β-mediated gene transcription downstream of the nuclear translocation of phosphorylated Smad3. A, primary NHLFs were treated with TGF-β (5 ng/ml) in the presence or absence of MVE or its control cation (TPP) (both 1 μm), and cytosolic and nuclear fractions were isolated and immunoblotted using antibodies to phosphorylated (p-Smad3) and total Smad3. Antibodies to actin and RNA polymerase II (RNA pol II) were used as loading controls and to ensure exclusion of cytosolic proteins. B, primary NHLFs were treated with TGF-β (5 ng/ml) with or without MVE or TPP (both 1 μm), and 24 h later, cell death was measured by propidium iodine staining. C and D, NHLFs were transfected with a plasmid containing SBE-luciferase and treated 24 h later with TGF-β (5 ng/ml) with or without MitoQ (C), MVE (D), or TPP (all 1 μm), and SBE-luciferase activity was measured 24 h later. E and F, primary NHLFs were grown to 70% confluence and incubated with TGF-β (5 ng/ml) with or without MitoQ (E), MVE (F), or TPP, and 24 h later, the levels of mRNAs encoding α-SMA, CTGF, and NOX4 were measured using quantitative RT-PCR in cell lysates. Error bars represent mean ± S.E. (n = 3 for all measures). *, p < 0.05 for comparison between TGF-β1 and the control; †, p < 0.05 for comparison between MitoQ or MVE and TPP.