Figure 6
NOX-4 expression and intracellular ROS production depended on c-JNK activity. A. Serum-starved RMF-EG cells were pretreated or not with 10 μM SP600125 for 16 h and stimulated for 24 h with 5 ng/ml of TGF-β1. NOX-4 protein expression was evaluated by western blot. B. Densitometric analysis of western blot bands in A. Columns represents mean ± SE ratio of NOX-4 to Actin signal of three independent experiments. C. Time-course of NOX-4 mRNA expression in TGF-β1-stimulated RMF-EG treated or not with SP600125 measured by qPCR. D. Time-course of intracellular ROS production evaluated by fluorescence generated with H2DCFDA incubation in TGF-β1-stimulated RMF-EG cells pre-treated or not with 10 μM SP600125. Values are representative of three separate experiments. E. RMF-EG cells were infected either with viral particles carrying a sequence specific shRNA targeting JNK1,2 (shJNK1,2) or a non-related sequence (shCTL) and treated or not with 5 ng/ml of TGF-β. α-SMA, NOX4 and JNK protein expression was evaluated by western blot. Figure is a representative result of an experiment performed in duplicate. F. Smad2/3 knocking down does not affect NOX4 expression. RMF-EG cells (3x105) transfected with a siRNA directed to Smad2/3 were stimulated with 2.5 ng/ml of TGF-β1 for 24 h and mRNA expression was analyzed by qPCR.