Figure 1. Constitutive activation of JAK2 and downstream pathways by MPLW515L enhance the chemotactic response of MO7e cells to CXCL12.

MO7e cells were transduced with retroviral vectors expressing GFP alone or GFP with MPLW515L gain-of-function mutation. A. Investigation by western-blot of MPL expression and downstream signaling pathway (STAT3, AKT and ERK) activation in MO7e- MPLW515L and mock-transduced cells (MO7e-GFP) in the cells without starvation. Treatment with AZD1480 (2 μM) inhibits the constitutive phosphorylation of STAT3, AKT and ERK. B. Effect of MPLW515L expression on CXCL12-induced migration in transwell assay. Migration assay data obtained for control MO7e cells were pooled with those obtained when TPO effects were tested (Figure 2A). C. MPL W515L expression in MO7e cells and AZD1480 (2 μM) inhibitor do not modify CXCR4 membrane expression. D. AZD1480 (2 μM), Ruxolitinib (2 μM) or BEZ235 (10 μM) strongly inhibited chemotaxis of mutant cells to CXCL12 (100 ng/ml). E. Effects of the dual PI3K/mTOR inhibitor BEZ235 on MO7e cell migration in response to CXCL12. MO7e cells were treated with various concentrations (1, 5 or 10 μM) of BEZ235 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. Treatment with 10 μM of BEZ235 inhibitor does not induce apoptosis in human MO7e cells. After treatment with the indicated concentrations of BEZ235 for 6 hours, MO7e cells were stained with Annexin-V and propidium iodide. The percentages of Annexin-V positive cells were determined by flow cytometry. G. Effects of BEZ235 inhibitor on the viability of MO7e cells. Columns represent the mean of 3 independent experiments. Data are represented as mean ± SEM.