Figure 5
In vitro treatment with SB-505124 abrogates TGF-β1-induced EMT and changes in E-cadherin expression, cell motility and cell mechanics. (A) The SB-505124 + TGF-β1-treated LLC group consisted of cells that were treated with 10 ng/ml TGF-β1 in the presence of 10 μM SB-505124 for 48 hr. Cell lysates were analyzed by western blot analysis with an anti-E-cadherin antibody. Equal loading of protein was verified by the analysis of GAPDH. This experiment was repeated three times with similar results. The data showed that TGF-β1-treated LLC cells expressed less E-cadherin than the untreated cells and the SB-505124 + TGF-β1 co-treated cells. E-cadherin was equally expressed in the latter two samples. (B) Phase contrast images of the wound-healing assay are shown. The migration of the cell monolayer was observed by time-lapse microscopy and photographed at 0, 8 and 24 h after wounding. Original magnification was 10×. The percentages of wound closure for the control, the SB-505124 + TGF-β1-treated and the TGF-β1-treated cells were calculated from the mean of four wound widths at each time point. The average closure rate was highest in TGF-β1-treated cells. (C) LLC cells were assessed with the MMS. TGF-β1-treated cells displayed significant differences in stiffness and adherence when compared with the control and the SB-505124 + TGF-β1 co-treated cells based on CS, TS and AF measurements. Scale bar = 100 μm. *, p < 0.05, **, p < 0.01, ***, p < 0.001.