Figure 2.

CB2 mediates TGF-β1-induced fibrogenic responses in vitro. (a, b) Over-expression of CB2 induced fibronectin expression in vitro. HKC-8 cells were transfected with CB2 expression vector (pCMV-CB2) or empty vector (pcDNA3) as indicated. Numbers (1 to 3) represent triplicate wells in a given group. Representative Western blot (a) and quantitative data (b) are presented. *P < 0.05 versus pcDNA3 group. (c, d) Western blotting analyses show that TGF-β1 dose-dependently induced CB2 expressions in cultured tubular epithelial cells in vitro. Human kidney proximal tubular cells (HKC-8) were incubated with different concentrations of TGF-β1 as indicated. Representative Western blot (c) and quantitative data (d) are presented. *P < 0.05 versus controls. (e–g) Knockdown of CB2 inhibited TGF-β1-induced fibrogenic responses in vitro. HKC-8 cells were transfected with CB2-specific siRNA or control siRNA, followed by incubation with TGF-β1 (2 ng/ml). Cell lysates were immunoblotted with antibodies against CB2, fibronectin, α-SMA and GAPDH, respectively. Representative Western blot (e) and quantitative data on the relative abundance of fibronectin (f) and α-SMA (g) proteins in different groups are presented. *P < 0.05 versus controls. †P < 0.05 versus control siRNA in the presence of TGF-β1. (h) Representative images show immunofluorescence staining of fibronectin in different groups. Arrow indicates positive staining. Scale bar, 50 μm. (i) Western blotting analyses show that TGF-β1-induced signal transduction was significantly attenuated by knockdown of CB2. Human kidney proximal tubular cells (HKC-8) were transfected with CB2 siRNA, then incubated with TGF-β1. Quantitative data (j through m) are presented. *P < 0.05 versus controls. †P < 0.05 versus control siRNA in the presence of TGF-β1.