Figure 4

Preventive Effect of RSK inhibitor SL0101 on MSP and MSP plus TGF-β1-induced EMT: M-RON cells (1 × 10⁵ cells/well in a 24-well plate) were incubated overnight and then stimulated with MSP (2 nM), TGF-β1 (5 ng/ml), or both at 37°C for 24 h. Small chemical inhibitors specific to RON (CP-1, 100 nM), Erk1/2 (PD98059, 100 μM), PI-3kinase (wortmannin, 50 μM), and RSK (SL0101, 50 μM) were added simultaneously. A) Cell morphological changes were observed and photographed using Olympus BK71 microscope equipped with CCD camera. Scale bars represent 20 μm. B) Cellular proteins (50 μg per sample) were also prepared for Western blot analysis. Expression of E-cadherin, claudin-1, and vimentin was determined by using specific antibodies. Membranes were also reprobed with antibodies to actin as the loading control. C) Transcription repressor Snail in nuclear proteins was detected by Western blot analysis using anti-Snail antibody. Preparation of nuclear factions was performed as previously described [45]. D) and E) β-catenin redistribution and F-actin reorganization was determined by immunofluorescent analysis [35] using antibodies specific to β-catenin and F-actin, respectively. Data shown here are from one of three experiments with similar results. Scale bars represent 10 μm.