Effect of HCV-induced TGF-β1, furin, and TSP-1 on hepatic stellate cells activation.

<p>A) Mock- and HCV-infected cells were transfected with siGFP, siTGF-β1, sifurin, and siTSP-1 as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056367#s2" target="_blank">Materials and Methods</a>. To determine the levels of silencing, total cellular RNA was extracted, and subjected to quantitative RT-PCR using furin, TSP-1, and TGF-β1 specific primers. The data shown here represent the means standard deviations of three independent experiments performed in triplicate. *denotes p<0.05 compared to mock cells. **denotes p<0.05 compared to HCV-infected mock-transfected cells. B) Equal amounts of cellular lysates from above siRNA transfected cells were subjected to western blot analysis using anti-TGF-β1, furin, and TSP-1 antibodies. Lane 1, Lysates from mock (Huh7.5) cells; lane 2, HCV-infected cells; lanes 3 and 4, HCV-infected cells transfected with siGFP or siTGF-β1, sifurin and siTSP-1. Actin and albumin were used as protein loading controls. C) HSC line, LX-2 was grown and serum starved for 48 h prior to incubation with conditioned medium (CM) collected from HCV-infected Huh-7.5 cells transfected with siGFP, siTGF-β1, sifurin, or siTSP-1 for 24 h. Total cellular RNA was extracted from LX-2 cells and the levels of α-SMA and Col 1 alpha 1 mRNA were determined by quantitative RT-PCR. The data shown here represent the means standard deviations of three independent experiments performed in triplicate. *denotes p<0.05 compared to LX-2 cells incubated with CM from mock cells. **denotes p<0.05 compared to LX-2 cells incubated with CM from HCV-infected cells. D) Cellular lysates from cells as described above were subjected to immunoblot analysis using antibodies against α-SMA and GAPDH. Lanes 1 and 2, lysates from LX-2 cells incubated with CM from mock- and HCV-infected cells; Lanes 3–6, lysates from LX-2 cells incubated with CM from HCV-infected cells transfected with siGFP, siTGF-β1, sifurin, and siTSP-1. Bottom panel shows the protein loading control immunoblotted with anti-GAPDH primary antibody.