Fig. 7.

Blockade of IL-17-mediated TGF-β associated pathways prevents EMT and col(V) expression in vitro. A: Western blotting analysis of α-SMA protein expression in RLE-6TN cells pretreated with pharmacological inhibitor against ALK5 In (SB431542; 2.5 μM; ALK5 In) for 1 h and then treated with IL-17 (100 ng/ml) or TGF-β (10 ng/ml) or both IL-17 and TGF-β for 72 h. B: α-SMA protein expression was determined by Western blotting in RLE-6TNs pretreated with pharmacological inhibitors against p38 MAPK (SB202190; 5 μM) and FAK (PP2; 5 μM) at the indicated doses for 1 h and then stimulated with IL-17A (100 ng/ml) for 72 h. TGF-β was used as the positive control. C: RLE-6TNs were treated as described in B and dual-labeled for E-CAD (red) and α-SMA (green) at 72 h. Using immunofluorescent labeling, Vimentin (green; at 48 h) and S100A4 (red; at 24 h) were detected. Nuclei were counterstained with DAPI. Images were captured at ×20 magnification. To further investigate col(V)-related signaling, RLE-6TNs were treated as described in A (Fig. 7D) and B (Fig. 7E). Conditioned media were normalized for equal protein concentration by loading 30 μg of the proteins for all samples and then immunoprobed for col(V).