Figure 7.

TGF-β1 induced prolidase-dependent increase in collagen type 1 expression in NIH3T3 fibroblasts.A and B, NIH3T3 fibroblasts were plated in 8-well chamber slides and serum-starved for 6 h and were treated with either vehicle (DMSO) or SIS3 (10 μM) 1 h prior to stimulation with TGF-β1 (5 ng/ml) for 6 h. A, representative confocal images of cells stained for prolidase (red) and Col1A1 (green) and the nuclei (blue). Scale bar, 20 μm. B, the graph represents the mean fluorescence intensity of prolidase and Col1A1 in SIS3-treated vs untreated cells in the presence and absence of TGF-β1. C–E, NIH3T3 cells were plated in 8-well chamber slides and transfected with either control siRNA or prolidase specific siRNA for 48 h. Following which the cells were serum starved and stimulated with TGF-β1 (5 ng/ml) for 6 h. After treatment the samples were prepared for confocal microscopy. C, representative immunoblot showing the knockdown of prolidase expression with siRNA, using β-actin as loading control. D, representative confocal images of prolidase and Col1A1 expression in NIH3T3 cells after staining. Scale bar, 20 μm. E, the graph represents the mean fluorescence intensity of prolidase and Col1A1 in siRNA-transfected vs control-siRNA-transfected cells in the presence and absence of TGF-β1. Data in (B and E) are mean values of three independent experiments with error bars representing SEM. ∗ p value of < 0.05 for the statistical comparison of (B) untreated control vs TGF-β1 treated samples and TGF-β1 treated vs TGF-β1 + SIS3 treated samples, and (E) untreated control vs TGF-β1 treated samples and TGF-β1 treated vs TGF-β1 + Prolidase siRNA samples.