Figure 2

Pharmacological properties of GMME1 on EG7 tumor cells. A. Following the confirmation that EG7 cells express CCR2 by RT-PCR, 10⁵ EG7 cells/well were cultured for 48 hrs in presence of increasing amounts of CCL2 5-76, CCL2 1-76 or GMME1 and the proliferative response measured by MTT. CCL2 5-76, and to a lesser extent, CCL2 1-76 were capable of inducing the proliferation of EG7 cells as opposed to GMME1 (P < 0.05; n = 6/group). B. Following the addition of 1.5 pmol of GMME1 on EG7 cells for 48 hrs (lower panel), a PI/Annexin-V co-staining demonstrates that GMME1 leads to apoptosis induction (32% dead cells). None of the B16 cells, which are CCR2 null, were affected by the addition of GMME1. C. EG7 cells cultured with GMME1 for 48 hrs induce de novo expression of the pro-apoptotic BAX protein. D. Following the stimulation of EG7 cells for different time points, cell lysate was analysis by a pSTAT3 ELISA (P < 0.05; n = 6/group). The experiments were repeated using the 5 min time point, then lysate was probed by WB. Total STAT3 was used as loading control. E. WT or CCR2^-/- monocytes were purified and cultured with 1.5 pmol of GMME1 for 48 hrs before a PI/Annexin-V co-staining. Even though 58% of WT monocytes died, no major cell death was detected with CCR2^-/- monocytes.