AML cells require mTOR for long-term survival in vitro. (A) Samples were thawed and immediately lysed for analysis. Samples were lysed and analyzed by SDS-PAGE and Western blotting for the indicated proteins. U937 cells treated with rapamycin (lane 1, +) or not treated were used as control samples. Note that in U937 samples, inhibition of phosphorylation of 4EBP-1 is not complete after treatment with rapamycin. (B) Samples were thawed, allowed to recover for 2 hours, counted, and plated at 2 × 10⁶ cells/mL in 96-well plates. Cells were incubated in EGM2 medium for 48 hours in indicted concentrations of rapamycin, and relative cell survival was measured using the XTT assay. Results were standardized so that untreated samples were assigned a value of 1.0. (C) Cells were processed and cultured as for panel B, but plates were incubated for 7 days before analysis by XTT assay. (D) Normal human CD34 cells were cultured for 2 or 7 days in serum-free medium with hematopoietic cytokines, and relative cell survival was measured using the XTT assay described under “Materials and methods.”