Figure 3.
PI3Kγ regulates the PPP of LSCs. (A) Kyoto Encyclopedia of Genes and Genomes pathway analyses were performed with transcriptome data of WT and Pik3cg-KO BM L-GMP cells at 4 weeks after primary transplantation (n = 3 mice per group). (B) Gene set enrichment analysis evaluating changes in the PPP in WT and Pik3cg-KO BM L-GMP cells at 4 weeks after primary transplantation. (C) mRNA levels of G6pd, Pgd, Tkt, and Taldo1 were compared by quantitative RT-PCR between WT and KO L-GMPs at 4 weeks after primary transplantation (n = 3). (D) Downregulated metabolic pathways in Pik3cg-KO L-GMP cells by metabolomics analysis at 4 weeks after primary transplantation. (E-F) The intensities of NADPH and NADP+ (E, n = 6) and the ratio of NADPH/NADP+ (F, n = 6) were evaluated in WT and KO BM L-GMP cells at 4 weeks after primary transplantation. (G) Flow cytometric analysis of ROS levels of WT and KO BM L-GMP cells at 4 weeks after primary transplantation (n = 5). (H) Flow cytometric analysis of ROS levels in WT and KO BM L-GMP cells upon 1 mM NAC treatment for 2 hours at 4 weeks after primary transplantation (n = 3). (I-J) Colony numbers (I) and derived total cell count (J) of WT and Pik3cg-KO BM L-GMPs upon 1 mM NAC treatment at 4 weeks after primary transplantation (n = 3). (K) The protein levels of p-AKT (S473), p-AKT (T308), AKT, G6PD, PGD, and PI3Kγ in WT and Pik3cg-KO BM L-GMP cells were measured by western blot at 4 weeks after primary transplantation. (L) The changes of metabolites in the PPP between WT and Pik3cg-KO BM L-GMP cells at 4 weeks after primary transplantation (n = 6). (M-N) The frequencies of leukemia cells in the PB (M, n = 5) 4 weeks post-transplantation and overall survival (N, n = 5) were compared among the recipients transplanted with WT, Pgd-overexpressing WT, Pik3cg-KO, and Pgd-overexpressing KO AML cells. (O-P) The metabolite levels were measured in WT, Pgd-overexpressing WT, Pik3cg-KO, and Pgd-overexpressing KO L-GMP cells and normalized against WT+vector cells 4 weeks after transplantation (n = 5). Data are represented as mean ± SEM. Student 2-tailed unpaired t test for panels C,F-G,L, 1-way ANOVA with Tukey multiple comparison test for panels H-J,M,P, 2-way ANOVA with Sidak multiple comparison test for panels E,O, and log-rank test for panel N were used for the comparison of statistical significance. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; n.s., not significant.

PI3Kγ regulates the PPP of LSCs. (A) Kyoto Encyclopedia of Genes and Genomes pathway analyses were performed with transcriptome data of WT and Pik3cg-KO BM L-GMP cells at 4 weeks after primary transplantation (n = 3 mice per group). (B) Gene set enrichment analysis evaluating changes in the PPP in WT and Pik3cg-KO BM L-GMP cells at 4 weeks after primary transplantation. (C) mRNA levels of G6pd, Pgd, Tkt, and Taldo1 were compared by quantitative RT-PCR between WT and KO L-GMPs at 4 weeks after primary transplantation (n = 3). (D) Downregulated metabolic pathways in Pik3cg-KO L-GMP cells by metabolomics analysis at 4 weeks after primary transplantation. (E-F) The intensities of NADPH and NADP+ (E, n = 6) and the ratio of NADPH/NADP+ (F, n = 6) were evaluated in WT and KO BM L-GMP cells at 4 weeks after primary transplantation. (G) Flow cytometric analysis of ROS levels of WT and KO BM L-GMP cells at 4 weeks after primary transplantation (n = 5). (H) Flow cytometric analysis of ROS levels in WT and KO BM L-GMP cells upon 1 mM NAC treatment for 2 hours at 4 weeks after primary transplantation (n = 3). (I-J) Colony numbers (I) and derived total cell count (J) of WT and Pik3cg-KO BM L-GMPs upon 1 mM NAC treatment at 4 weeks after primary transplantation (n = 3). (K) The protein levels of p-AKT (S473), p-AKT (T308), AKT, G6PD, PGD, and PI3Kγ in WT and Pik3cg-KO BM L-GMP cells were measured by western blot at 4 weeks after primary transplantation. (L) The changes of metabolites in the PPP between WT and Pik3cg-KO BM L-GMP cells at 4 weeks after primary transplantation (n = 6). (M-N) The frequencies of leukemia cells in the PB (M, n = 5) 4 weeks post-transplantation and overall survival (N, n = 5) were compared among the recipients transplanted with WT, Pgd-overexpressing WT, Pik3cg-KO, and Pgd-overexpressing KO AML cells. (O-P) The metabolite levels were measured in WT, Pgd-overexpressing WT, Pik3cg-KO, and Pgd-overexpressing KO L-GMP cells and normalized against WT+vector cells 4 weeks after transplantation (n = 5). Data are represented as mean ± SEM. Student 2-tailed unpaired t test for panels C,F-G,L, 1-way ANOVA with Tukey multiple comparison test for panels H-J,M,P, 2-way ANOVA with Sidak multiple comparison test for panels E,O, and log-rank test for panel N were used for the comparison of statistical significance. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; n.s., not significant.

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