PP242-induced apoptosis in primary AML cells. (A) Nine samples obtained from patients diagnosed AML with high blast count were treated with PP242 at indicated concentrations in the presence or absence of MS-5 stromal cells for 48 hours. Apoptotic cells were detected using annexin V⁺ flow cytometry. The percentage of specific annexin V⁺ apoptosis was calculated as described in “Cell viability.” Data represent average ± SEM of specific apoptosis in 9 AML samples. (B) Comparison of proapoptotic effects of PP242 and temsirolimus. OCI-AML3 cells (top panel) and 2 primary AML samples (bottom panel) were treated with PP242 and temsirolimus with or without MS-5 coculture for 72 hours, and the percentage of apoptotic cells (annexin V⁺/DAPI⁺/CD45⁺) was detected by flow cytometry. (C) AML cells from samples of patient no. 1 and no. 2 cocultured with MS-5 were treated with 2.5μM PP242 for 24 hours. Cells were harvested, and lysates subjected to immunoblotting with indicated antibodies to probe mTOR signaling.