Akt/mTORC1 hyperactivity in Itpkb^−/− HSC. (A-B) WT (solid) or Itpkb^−/− (hatched) LSK cells were treated in vitro with medium or SCF ± cell-permeable IP₄/PM or Akt-inhibitor VIII (Akt-I) and analyzed by FACS for T₃₀₈-phosphorylated Akt (pAkt_T308) content. (A) Representative FACS data from 4 independent experiments. (B) pAkt_T308 median fluorescence intensity (MFI) in WT (black squares) or Itpkb^−/− (gray circles) LSK cells, normalized to the MFI of unstimulated WT cells (n = 4). Statistical significance of genotype differences was determined by unpaired Student t test. (C) Total Akt protein content in WT or Itpkb^−/− LSK cells. Shaded histogram, isotype control. (D) WT (solid) or Itpkb^−/− (hatched) LSK cells treated as in (A) were analyzed for pAkt_T308 and phospho-ribosomal protein S6 (pS6_S235/S236) content in LSK CD34⁻CD48⁻CD150⁺ LT-HSC, and MPP1-3 subpopulations. (E) Itpkb^−/− or WT LT-HSC enriched GSEA gene sets associated with Akt/mTOR signaling and metabolic activation were identified as in Figure 3C. Gene set descriptions are shown in supplemental Table 5.