Leukemic stem cells may require PI3 kinase for survival. Leukemic patient cells were thawed and rested for 2 hours in serum-free medium. Viable cells were counted and divided into 4 groups. One group of cells was injected immediately into NOD/SCID animals, with 5 × 10⁶ mononuclear cells injected per animal (fresh cells). The 3 remaining groups were incubated overnight in serum-free medium containing cytokines and either nothing additional (not shown), 0.1% DMSO, or LY294002 at 25 μg/mL in DMSO. Cells from these 3 groups were injected into irradiated NOD/SCID animals after a 16-hour incubation. Animals were observed for 7 weeks, humanely killed, and analyzed for engraftment. Total bone marrow cells were harvested, red blood cells removed, and remaining cells stained with antibodies to human CD45 (y-axis) and human CD33 (x-axis). Corresponding stains for human CD19 were negative. (A-D) Contour plots of engraftment of CD45⁺/CD33⁺ cells in a representative experiment. Engrafted cells are in the upper right quadrant of each panel, and percentage of human cells is indicated by inset number. (E) Graph of individual results and averages (medians indicated by horizontal bars) from 4 separate animals in each group. Each point represents a single animal. Averages are average engraftment for the 4 animals in each cell treatment group.