Loss of HSC quiescence in Itpkb^−/− mice. (A) Annexin V/7-AAD stain of the indicated phenotypic LT-HSC or MPP subsets in WT or Itpkb^−/− BM. Numbers denote percent of cells per quadrant. Viable cells are double-negative. Representative of 2 independent experiments (n_wt = 3; n_KO =4). (B-C) Cell-cycle status of LSK Flk-2⁻ LT-HSC/MPP1-3 and LSK CD34⁻ LT-HSC in WT or Itpkb^−/− mice, analyzed by Hoechst/Ki67 stain. (B) Representative data from 8 independent experiments. G₀, Ki67⁻Hoechst 33342⁻. G₁, Ki67⁺Hoechst 33342⁻. S, Ki67⁺Hoechst 33342^int. G₂/M, Ki67⁺Hoechst 33342^hi. Numbers denote percent of cells per gate. (C) Percent of LT-HSC per cell-cycle phase for 11 mice/genotype (horizontal bars, means). KO, Itpkb−/−; WT, wild type. Statistical significance was determined by paired Student t test. (D) In vivo BrdU incorporation in LSK CD34⁻ LT-HSC in WT or Itpkb^−/− mice during a 2-hour pulse. Numbers denote percent of BrdU⁺ cells. BrdU⁻, uninjected WT control. Representative of 2 independent experiments. (E-F) Forward (FSC-A)/side-scatter (SSC-A) analysis of LSK CD34⁻CD150⁺ LT-HSC, LSK CD34⁺Flk-2⁻ MPP1-3, and LSK CD34⁺Flk-2⁺ MPP4 in WT or Itpkb^−/− BM. (E) Representative data from 5 independent experiments. Numbers denote percent of cells per gate. (F) Pooled data with means (horizontal lines) and P values for the indicated comparisons (unpaired t test; n = 11).