Figure 5

Inhibition of c-JNK activity reduced α-SMA/CTGF expression and partially reduced TGF-β-dependent transcriptional activity. A. CTGF and α-SMA mRNA time-course expression in RMF-EG cells. Serum-deprived RMF-EG cells were pre-treated or not with 10 μM SP600125 for 16 h and further stimulated with 5 ng/ml of TGF-β1 at the indicated times. mRNA expression of both genes were evaluated by qPCR. B. RMF-EG cells were treated with TGF-β1 (5 ng/ml) in the presence or absence of 10 μM SP600125. Protein expression of CTGF and α-SMA was evaluated by western blot after 24 h and 72 h respectively. C. Densitometric analysis of western blot bands in B. Columns represents mean ± SE ratio of α-SMA to Tubulin and CTGF to Actin signal from three independent experiments. D. RMF-EG cells were transiently transfected with 3TP-luc luciferase construct and then pre-treated or not with SP600125 before stimulation with TGF-β1. Values presented are representative of three separate experiments performed in triplicate. E. Immunofluorescence analysis to α-SMA of control and TGF-β1-treated (48 h) RMF-EG cells preincubated in the presence or absence of DPI (2,5 μM) or SP600125 (10 μM). Figure is a representative image of an experiment performed in duplicate.