Figure 6
Regulatory effect of RON160 and RONE5/6inon EMT-like activities in MDCK cells: A) Effect of RON, RON160, and RONE5/6in on epithelial/mesenchymal protein expression. Proteins (50 μg per sample) from cell lysates prepared after 72 h incubation were subjected to Western blot analysis using antibodies specific to vimentin and E-cadherin, respectively. Actin was used as the loading control. B) Effect of RON, RON160 and RONE5/6in on cell morphological changes. MDCK, M-RON, M-RON160 and M-RONE5/6in cells were cultured for 24 h and then stimulated with 2 nM of MSP for 48 h. Cell morphological changes were observed under Olympus Inverted microscope and photographed. C) Effect of RON, RON160 and RONE5/6in on spontaneous or MSP-induced MDCK cell migration. Cell monolayer was wounded as previously described [29] and stimulated with or without 2 nM of MSP for 16 h. Chemical inhibitors such as PD98059 (10 nM, specific to MAP kinase) and wortmannin (50 μg/ml, specific to PI-3 kinase) were added simultaneously. The wounded area covered by migrated cells was measured and shown as % of the covered space. Data shown here are from one of three experiments with similar results.