Results in Figure 3B demonstrated that MSP in combination with TGF b1 induced RSK2 nuclear translocation and phosphoryla this tion. This effect was accompanied by Erk1/2 phosphory lation. A major difference was that the time course for both RSK2 and Erk1/2 phosphorylation lasted longer in MSP and TGF b1 co stimulated cells than in cell treated with MSP alone. We further validated results from Western blotting by studying cellular RSK and Erk1/2 distribution using DSU confocal microscope image analysis. Cytoplasmic and nuclear RSK2 and Erk1/2 were detected by anti RSK2 or Erk1/2 immunofluorescent analysis. As shown in Figure 3C, RSK2 immunofluorescent staining was detected in both cytoplasmic and nuclear compartments in control M RON cells.

Upon MSP stimulation, increased nuclear fluorescent intensity was observed, indicating nuclear accumulation of RSK2 and Erk1/2. We noticed that RSK2 nuclear Inhibitors,Modulators,Libraries staining appeared as a pattern of condensed granules. Cellular distribution of Erk1/2 in control cells was similar to that of RSK2. MSP induced Erk1/2 nuclear translocation with increased nuclear fluorescent Inhibitors,Modulators,Libraries intensity. The patterns of Erk1/2 nuclear staining were in a relatively diffused manner. Consistent with these observations, RSK 2 nuclear accu mulation also was observed in cells stimulated with MSP plus TGF b1 with granule like staining pattern. Again, Erk1/2 Inhibitors,Modulators,Libraries accumulated in nucleus with combined stimulation but distributed in a more diffused pattern. These results, together with those in Figure 3A and 3B, demonstrated that distribution and phosphorylation between RSK2 and Erk1/2 upon MSP stimulation exist.

Preventive effect of RSK2 inhibitor SL0101 on MSP or MSP plus TGF b1 induced EMT To determine if RSK2 is indeed an effector molecule, Inhibitors,Modulators,Libraries we studied the effect of SL0101 on MSP induced EMT. We also used TGF b1 to induce EMT for evaluation. Results in Figure 4A showed that MSP induced spindle like morphological changes in M RON cells. As expected, this effect Inhibitors,Modulators,Libraries was prevented by CP 1 and PD98059, but not by PI 3 kinase inhibitor wortmannin. Consistent with results shown in Table 1, SL0101 significantly prevented MSP induced spindle like morphology. SL0101 also pre vented TGF b1 induced cell shape changes, but its effect was not complete. Moreover, the synergistic effect of MSP and TGF b1 in cell morphology was affected by SL0101. In all these cases, altered cell mor phology was significantly restored to original epithelial appearance. Experiments were then conducted to determine if SL0101 regulates E cadherin, claudin 1, selleck Carfilzomib and vimentin expression. CP 1, PD98059, and wortmannin were included as controls. SL0101 completely prevented MSP induced reduction of E cadherin. Sl0101 also pre vented increased vimentin expression.