THPO/MPL signaling enhances the growth and survival of CD41⁺ Evi1 leukemia cells. (A) Proliferation assays of CD41⁺ and CD41⁻ BM cells from Evi1 leukemia mice using OP9 coculture system. CD41⁺ or CD41⁻ cells (5 × 10⁴ cells per well) were seeded onto a confluent layer of OP9 stromal cells in the presence of SCF and THPO, THPO alone, or SCF alone. After 7 days of culture, cells were harvested by trypsinization and the number of viable leukemia cells was counted. Error bars indicate SD (n = 7; **P < .01, ***P < .001, Tukey’s test). (B-C) The antiproliferation effect of an anti-Mpl antibody against CD41⁺ cells. CD41⁺ (B; n = 4) or CD41⁻ (C; n = 3) cells were seeded onto OP9 stromal cells with or without THPO, or THPO with an anti-Mpl antibody (100 ng/mL), and cultured for 7 days. The number of viable cells was determined as described in panel A. Error bars indicate SD (*P < .05, Dunnett’s test). (D) Apoptosis analysis of CD41⁺ and CD41⁻ cells cocultured with OP9 cells. CD41⁺ or CD41⁻ cells were cultured in the same condition as panel A. After 7 days culture, cells were harvested and stained with Annexin V, followed by FACS analysis. Error bars indicate SD (n = 3; **P < .01, Tukey’s test). (E) BM- and SP-MNCs of Evi1 leukemia mice were serum-starved in α-MEM containing 1% bovine serum albumin (BSA) for 60 minutes and then stimulated with SCF or THPO in α-MEM containing 0.1% BSA for 60 minutes. Unstimulated cells were used as negative controls. The phosphorylation levels of STAT3, STAT5, ERK1/2, and AKT were analyzed by western blotting. (F-G) CD41⁺ cells were treated with dimethylsulfoxide (DMSO) as a vehicle control, a JAK2 inhibitor (AG490; 20 μM), or an MEK inhibitor (PD98059; 20 μM) on OP9 stromal cells in the presence of THPO for 7 days. (F) The number of viable cells was counted. Error bars indicate SD (n = 3; Dunnett’s test). (G) The rate of apoptotic cells was determined by FACS. Error bars indicate SD (n = 4; *P < .05, Dunnett’s test). The concentration of the cytokines used in these experiments was 50 ng/mL.