PP242-mediated intracellular mTOR signaling in primary AML cells and MSCs cultured alone or cocultured. Measurement of the MFI of intracellular p4EBP1 (Thr37/46; A), pAKT (Ser473; C), and pS6K (Ser235/236; B) in primary AML cells, AML CD34⁺ progenitor cells, and primary AML MSCs (D) cultured alone and in cocultures using flow cytometry, The gray bars represent the MFI of the phosphorylated proteins before, and the black bars after treatment with PP242 for 72 hours. Cells were cultured in medium only (“alone”), or cocultured with MSCs (“floating” or “attached” cells, respectively). Data represent average ± SEM of MFI of the phospho-proteins measured in the indicated number of primary samples. Paired 2-sample t test was used to determine reported P values. (E) Primary AML cells and MSCs in a transwell setting (described in “Leukemia/MSC/MS-5 coculture”) were treated with PP242 for 24 hours. Cells were then lysed, and mTOR signaling targets in the AML cells were detected by immunoblotting.