Figure 4

Pharmacological properties of GMME1 on human U266 tumor cells. A. U266 cells were analyzed by flow cytometry and were negative for the expression of CD19 while CD138 and CCR2 were detected. B. 10⁵ U266 cells were cultured with increasing amounts of CCL2 5-76, CCL2 1-76 or GMME1 and the proliferative response measured by MTT. CCL2 5-76 was capable of inducing U266 proliferation whereas GMME1 completely suppressed the proliferative response (P < 0.05; n = 6/group). C. Using 1.5 pmol of GMME1 on U266 cells for 48 hrs, a PI/Annexin-V co-staining demonstrates that GMME1 leads to apoptosis (42% cell death). D. A similar set-up was used for the assessment of STAT3 activation on U266 cells. Following the stimulation of U266 cells using different time points, cell lysate was analysis by a pSTAT3 ELISA. Since STAT3 is inhibited as of 10 min following GMME1 addition on U266 cells, the experiment was repeated at this time point then the lysate was probed by WB. Total STAT3 was used as loading control. To further confirm the inhibitory effect of GMME1 on these cells, the U266 conditioned-media was collected following 48 hrs post-treatment with the different test conditions and analyzed using a human IL-6 ELISA kit. No detectable levels of human IL6 could be observed in the GMME1 group as opposed to the remaining test conditions (*P < 0.05; n = 6/group).