AC220 promotes quiescence and a marked reduction of Flt3⁺ LSK cells, which is followed by a proliferative recovery response that parallels the induction of Flt3 ligand. (A) B6.CD45.1 mice repopulated with c-Cbl^A/− BM were dosed daily with vehicle or 10 mg/kg of AC220, and the BM cells were analyzed by flow cytometry at the indicated time points. The Flt3 profiles are of LSK cells, and the percentages of Flt3⁺ LSK cells (ie, MPPs) from each AC220-treated mouse is shown in red. (B) The gated Flt3⁺ LSK cells in panel A were analyzed for expression of Ki-67 and cell size (forward light scatter, FSC). The percentage of cells in each quadrant is shown. Cells in the top right quadrant are large Ki-67⁺ cells (ie, highly proliferative cells), and the cells in the lower left quadrant are small Ki-67⁻ cells (ie, G₀ quiescent cells). The profiles in panels A and B are representative of 3 vehicle- and 3 AC220-treated mice examined at each of the time points. (C-E) Data from 3 vehicle- and 3 AC220-treated mice examined at each time point showing the percentage of Ki-67⁺ MPPs (C), the geometric mean of forward light scatter (FSC) of MPPs (D), and the percentage of MPPs in the LSK population (E). (F) AC220 shows no effect in promoting apoptosis in MPPs. BM cells were stained with annexin V–FITC and propidium iodide (PI) and gated on the Flt3⁺ LSK population. (G) AC220 dosing promotes the induction of Flt3 ligand. Mice dosed daily with AC220 or vehicle were bled by cardiac puncture at the indicated times and their serum assayed for Flt3 ligand levels by ELISA. The data are from 3-7 mice at each time point. (H) The induction of phospho-S6 in MPPs parallels the increase in Flt3 ligand. The levels of intracellular phospho-S6 in gated Flt3⁺ LSK cells were determined by flow cytometry. The data are from 3 vehicle- and 3 AC220-mice at each time point. Data are means ± SEM. *P < .05; **P < .01; ***P < .001 using unpaired Student t test.