Figure 2

LPS/TLR4 axis affects Smad-mediated canonical TGF-β signalling pathway to drive EMT.

BPH-1 cells were incubated with LPS (100 ng/ml) and TGF-β1 (0.1 ng/ml) in the absence or presence of the TLR4 inhibitor CLI-095 (3 μmol/L). (A,B) Total RNA was subjected to qPCR. (C) Whole cell lysates were examined for E-cadherin and vimentin protein levels by western blot analysis. Values shown indicate relative levels corrected for β-actin. (D) BPH-1 cells were fixed, incubated with LPS and TGF-β1 (0.1 ng/ml) in the absence or presence of CLI-095 (3 μmol/L) and examined by immunocytochemical staining. Scale bar, 50 μm. (E) BPH-1 cells were incubated with LPS and TGF-β1 (0.1 ng/ml) in the absence or presence of CLI-095. Whole cell lysates were examined by using western blot analysis for p-Smad2/3 protein levels. (F) BPH-1 cells were fixed, incubated with LPS and TGF-β1 (0.1 ng/ml) in the absence or presence of CLI-095 and examined by immunocytochemical staining. Scale bar, 50 μm. *P < 0.05, determined by multifactorial ANOVA.