Requirement of RSK2 expression in MSP and TGF-β1 induced EMT-like activity in cancer cells. A) Expression of RON, RSK1, RSK2, and TGF-β receptors in human cancer L3.6pl and HT-29 cells. Cell lysates (50 μg/sample) were subjected to Western blot analysis using antibodies specific to individual proteins. B) MSP induces EMT-like activities in pancreatic cancer L3.6pl cells. L3.6pl cells (0.5 × 106 cells per dish) were cultured overnight and then stimulated at 37°C with MSP (2 nM), TGF-β1 (5 ng/ml), or both for 24 h. Cell morphological changes were observed by Olympus microscope and photographed with CCD camera. E-cadherin and vimentin expression was determined by Western blot analysis using cell lysates as described previously . Actin was used as the loading control. Scale bars represent 20 μm. C) Forced RSK2 expression facilitates MSP and TGF-1-induced EMT-like activity in HT-29 cells. Cells (2 × 106 cells per dish) were transiently transfected with 3 μg of pRKS2 plasmid or control vector pcDNA3.1 for 48 h and then stimulated with MSP and TGF-β1 as described above. Morphological changes and expression of individual proteins were determined as described in A. Scale bars represent 20 μM. D) RSK2 expression diminishes E-cadherin expression and increases vimentin expression. HT-29 cells were transiently transfected with pRSK2 plasmid for 48 h followed by stimulation with MSP, TGF-β1 or both for 24 h. Cell lysates were subjected to Western blot analysis using antibodies specific to E-cadherin or vimentin. β-actin was used as the loading control. Data shown here are from one of two experiments with similar results.