Figure 2. Cytokine-mediated activation of signaling pathways enhances the chemotactic response of MO7e cells to CXCL12.

A. Effect of TPO on CXCL12-mediated chemotaxis in MO7e-GFP control cells. After starvation, cells were treated or not with 10 ng/mL TPO and chemotaxis was performed in the presence of different concentrations of CXCL12. Migration assay data obtained for control MO7e-GFP cells were pooled with those obtained when the effect of MPLW515L was tested (Figure 1B). B. Investigation by Western-blot of AKT and ERK activation by CXCL12 and TPO. Cells were treated or not with TPO in the presence of different concentrations of CXCL12 for 10 minutes before lysate preparation. C. Effects of Ruxolitinib and BEZ235 on CXCL12-induced migration of MO7e cells upon stimulation with IL-6 (10 ng/mL), TPO (10 ng/mL) or SCF (25 ng/mL). Chemotaxis was assayed in the absence or presence of CXCL12 (100 ng/mL). D. Phosphorylation of STAT3, AKT and ERK in MO7e pretreated or not with Ruxolitinib (2 μM) or BEZ235 (10 μM) before stimulation with CXCL12 (100 ng/mL) alone, cytokines alone (IL-6, TPO, SCF) or combinations of both for 10 minutes. Cells were serum and cytokine starved for 4 hours prior cytokine addition. E. Effects of UO126 inhibitor treatment on MO7e cell migration in response to CXCL12.MO7e cells were starved and treated with various concentrations (1, 5, 10 or 30 μM) of UO126 for 2 hours. TPO (10 ng/mL) was then added or not and the migration in response to CXCL12 (100 ng/mL) was measured in chemotactic assay. The data shown represent the mean ± SEM of 3 independent experiments. F. Investigation by Western-blot of AKT and ERK activation by CXCL12 alone, TPO alone or combination of both in MO7e cells, after pre-incubation with various concentrations of UO126.