Leukemic cells expressing R1E and Mpl are sensitive to THPO signaling through Jak2/Pi3k/Akt. (A) Western blot analysis of signaling proteins activated by Mpl. MIG-R1E/MID (not expressing Mpl) and MIG-R1E/MID-MPL leukemic cells were stimulated with 0, 1, 2.5, 5, and 10 ng/mL THPO after serum starvation. Expression of phospho-Jak2, Jak2, phospho-Stat5, Stat5, phospho-Akt1, Akt1, phospo-Erk1/2, and Erk1/2 was tested by immunoblot analysis. (B) Apoptosis analysis (annexin V(+), 7-AAD-) of MIG-R1E/MID-MPL and MIG-R1E/MID leukemic cells estimated after 48-hour treatment with PBS (black), Thpo (gray), or Thpo and rapamycin (white); P < .01 (*), Student t test). (C) Apoptosis analysis (annexin V(+), 7-AAD-) of MIG-R1E/MID-MPL leukemic cells estimated after 48-hour treatment with PBS (white) or Thpo (black) with pretreatment of no inhibitor (none) or inhibitors for mTor (rapamycin), PI3K (wortmanin), Jak2 (TG101348), and MEK (PD98059). Experiments were performed in quadruplicate; P < .001 (*), Student t test. (D) Cell-cycle analysis (propidium-iodine staining) of MIG-R1E9a/MID-MPL treated as in panel C; subG1 (gray), G0/1 cells (dark gray), S phase cells (white), and G₂/M cells (black); P < .001 Student t test), (*). (E) Proliferation assays of MIG-R1E/MID-MPL leukemic cells after 48-hour culture with IL-3 (6 ng/mL), IL-6 (1 ng/mL), SCF (10 μg/mL), Thpo (20 ng/mL), or the combination of cytokines; each in triplicate. P < .001 (*), Student t test. (F) Kaplan-Meier survival curve of transplantations of MIG-R1E9a/MID-PL2 (left) and MIG-R1E9a/MID (right) leukemic cells in mice injected with mTOR inhibitor rapamycin (solid line, n = 8) or vehicle (dashed line, n = 8). P < .0001 log-rank test.