Effect of siRNA-mediated RSK2 knockdown on MSP and TGF-β1-induced EMT-like activities and cell migration by L3.6pl cells: A) Knockdown of RSK2 expression by specific siRNA restored E-cadherin expression and prevented vimentin induction. L3.6pl cells (2 × 106 cells per dish) were cultured overnight and transiently transfected with 3 μg/dish of pcDNA3.1, pRSK1, or pRSK2 by using lipofectamine (Invitrogen). Transfected cells were cultured for 72 h and then lysed with lysis buffer. Cellular proteins (50 μg per sample) were subjected to Western blot analysis as described previously using antibodies specific to RSK1, RSK2, E-cadherin, and vimentin, respectively. The membranes were also reprobed with rabbit IgG to actin as the loading control. B) L3.6pl cells were transiently transfected with pcRSK1 or pcRSK2 for 48 h followed by stimulation with MSP, TGF-β1, or both as described above. The wound healing assay was performed after a 24 h-incubation to determine the levels of cell migration. The percentage of wounded area covered by migrated cells was determined as previously described . Data shown here are from one of three experiments with similar results. Scale bars represent 50 μm.