The mTOR hyperactivity contributes to the hyperproliferation of Itpkb^−/− HSC. (A-C) Rapamycin (Rapa) injection can reverse the mTOR hyperactivation in, and hyperproliferation of LT-HSC and MPP in Itpkb^−/− mice with little effect on LT-HSC and MPP numbers. WT or Itpkb^−/− mice were intraperitoneally injected with rapamycin or vehicle every other day for 10 days, followed by FACS analysis of pS6_S235/S236 content (A) (n = 2), cell-cycle phase distribution (B) (Hoechst stain, n = 2), and total numbers (C) (n = 2) of LT-HSC and MPP subsets. Total Akt protein content was determined as a loading control. Bar graphs show mean percent of cells per indicated cell-cycle phase (B) or mean cell numbers for the indicated population (C). Error bars denote standard error of the mean. Representative of 2 (A) or 3 (B-C) independent experiments. Representative FACS data for (B) with gates are shown in supplemental Figure 7. In (A), vehicle injected WT and Itpkb^−/− mice, and rapamycin-injected Itpkb^−/− mice are represented by green, blue, and red open histograms, respectively. Gray solid histograms, isotype controls. In (B-C) these mice are represented by black solid bars, gray solid bars, and open bars, respectively. (D) Itpkb^−/− LT-HSC have reduced in vitro CFU activity and reduced sensitivity to rapamycin. Each 40 sorted WT (black solid bars) or Itpkb^−/− (open bars) LT-HSC were incubated in separate wells in M3434 methylcellulose media containing the indicated amounts of rapamycin (Rapa), vehicle, or nothing. Shown are mean numbers of colonies per well on day 7 (n = 3). Error bars denote standard error of the mean. Representative of 3 independent experiments.