Figure 9.

MitoTEMPO reduces TGF-β-induced mitochondrial ROS and expression of fibronectin and α-smooth muscle actin. (A,B) HLFs grown on 35 mm glass-bottom dishes were treated with the mitochondrial reactive oxygen species (mtROS) scavenger, MitoTEMPO (10 µM), for 1 h prior to transforming growth factor beta (TGF-β) (5 ng/mL) challenge for 24 h. After 24 h, cells were treated with MitoSOX for 30 min and mtROS was quantified using confocal microscopy. Cells (20–30/area) from at least 5–8 different areas of three independent experiments in triplicate were analyzed and quantified. (C) HLFs on 35 mm dishes were pre-treated with MitoTEMPO (10 µM) for 1 h prior to treatment with TGF-β (5 ng/mL) for 48 h. Cell lysates were analyzed for fibronectin (FN) and α-smooth muscle actin (α-SMA) expression by Western blotting. Shown is a representative blot from three independent experiments. (D) Quantification of FN (C), and (E) quantification of α-SMA from (C) was carried out by densitometry and Image J analysis. ** p < 0.001, and *** p < 0.0001 significantly different compared to control. MitoTEMPO decreased the TGF-β-induced mtROS, and FN and α-SMA expression. Scale bar: 20 μm.