Genetic ablation of Pik3cg in AML depletes LSCs. (A) Representative of flow cytometric analysis for WT and KO L-GMPs (Lin⁻CD127⁻Sca-1⁻c-Kit⁺CD34⁺CD16/32⁺) among the total live BM cells from recipients at 6 weeks after primary transplantation. (B) Quantification of the frequency of L-GMPs of the recipients in panel A (n = 5). (C) Quantification of the frequency of L-GMPs among the total live BM cells from moribund mice upon secondary transplantation (n = 5). (D) Representative images of colony formation of sorted WT and Pik3cg-KO BM L-GMPs during first and second plating. Scale bar, 20 μm. (E-F) Colony numbers (E) and derived total cell count (F) of sorted WT and Pik3cg-KO BM L-GMPs during the first and second plating were calculated (n = 3). (G) Survival curve of recipient mice transplanted with 100 L-GMP cells from MLL-AF9⁺ WT or Pik3cg-KO BM cells after primary transplantation (n = 10). (H-I) Limiting dilution assays for the frequency of the functional LSCs of WT and Pik3cg-KO BM cells. Different doses of GFP⁺ leukemia cells purified from primary recipients were transplanted into lethally irradiated recipients and the competitive repopulating units (CRUs) were determined using L-Calc software. (J-K) Gene set enrichment analyses evaluating changes in leukemia initiation/maintenance and myeloid differentiation gene signatures in WT and Pik3cg-KO BM L-GMP cells at 4 weeks after primary transplantation. Data are represented as mean ± SEM. Student 2-tailed unpaired t test for panels B-C,E-F and log-rank test for panel G were used for the comparison of statistical significance. ∗∗∗P < .001. FDR q-val, false-discovery rate q-value; NES, normalized enrichment score.