Fig. 4
Effects of U0126 on h-TGF-β1-induced EMT and invasion of KKU-M213 cells. Cells were treated with 5 ng/mL h-TGF-β1 and/or 10 µM SB431542 or 1 µM U0126 in 0.1% FBS media for 24 h or for the indicated time before assay. Cell lysates were analyzed for levels of total ERK1/2 (tERK) (a), phospho-ERK1/2 (pERK) (a, b), vimentin (b) phospho-Smad and Slug (d) by immunoblotting (see Additional file 1: Figure 1 for quantitative data). Conditioned media were analyzed for MMP-9 by gelatin zymography (c). Immunoblot and zymography data are representative of three independent experiments. E-cadherin localization was examined by immunofluorescence staining with anti-E-cadherin antibody (green) and DAPI (blue), which show that U0126 reversed the effect of TGF-β induced reduction of E-cadherin membrane localization (e). For the invasion assay, cells (105) in the same media as those of pre-treated condition were plated onto in vitro invasion Transwell chamber and allowed to invade for 6 h (f). Data are presented as mean ± SEM of percent change in number of invaded cells compared to h-TGF-β-treated condition obtained from three independent experiments. *P < 0.05, **P < 0.001