Fig. 6. The P13K/AKT pathway is involved in the TPO-mediated survival effect observed in clones with low levels of Mpl expression. (A) Proliferation assays. Cells of clone A were washed three times and left cytokine-deprived or incubated for 24 hours with 10 ng/ml of TPO, solvent alone (0.14% DMSO), or the indicated concentration of inhibitors. Cell proliferation was quantitated by [³H]thymidine incorporation. The results shown are means±s.d. of triplicate experiments. (B) Western blot analysis of STAT-5, ERK and AKT activation. Cells from clone A were washed three times, deprived of cytokines for 3 hours and stimulated with 50 ng/ml of TPO for 15 minutes, DMSO (inhibitor solvent) alone, 50 µM of the MEK inhibitor PD98059 or 10 µM of the P13K inhibitor LY294002. Activation of STAT5, ERK and AKT was analyzed as described in Fig. 1. ns, no stimulation. (C) Apoptosis analysis. Cells were washed three times and left cytokine-deprived or stimulated for 22 hours with 10 ng/ml of TPO, solvent alone (0.14% DMSO), or the indicated concentration of inhibitors. Cells were then labeled with PI and percentages of apoptotic cells were determined by flow cytometry. Results are representative of two independent experiments.

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