(A) Relative frequency of hematopoietic colonies formed by Pten^+/+ and Pten^S398A/S398A bone marrow cells in M3434 methylcellulose medium supplemented or not with 1 mM N-acetyl-L-cysteine (NAC) or 1 μM buthionine sulfoximine (BSO). n = 6 mice per genotype. Significance symbols indicate differences in the total number of colonies. CFU-GM, granulocyte/macrophage colony-forming unit; BFU-E, burst-forming unit, erythroid; CFU-GEMM, granulocyte/erythrocyte/monocyte/megakaryocyte colony-forming unit. (B) Proportion of lineage-negative (Lin^–), cKit-positive cells, assessed by flow cytometry, in methylcellulose colonies from the experiments shown in panel A. Each symbol represents a culture from an individual animal. (C) Diagram of the experimental strategy to evaluate the effect of NAC treatment on competitive hematopoietic reconstitution by Pten^+/+ and Pten^S398A/S398A bone marrow cells. (D) Proportion of circulating total blood cells, B220⁺ cells, CD3⁺ cells, and CD11b⁺ cells expressing the CD45.2 marker in the peripheral blood of lethally irradiated mice transplanted as depicted in panel C. Transplanted cells were pooled from 3 donor animals per genotype; “n” indicates the number of transplanted mice. Statistical analyses indicate differences at 20 weeks. (E) Flow cytometry plots depicting dihydrorhodamine (DHR) fluorescence in Lin^–cKit⁺Sca1⁺CD48^–CD150⁺PROCR⁺ cells expressing CD45.1 (wild-type) or CD45.2 (Pten^+/+ or Pten^S398A/S398A) isolated at 20 weeks after transplant. (F) Quantification of the data shown in panel E, expressed as a ratio of the DHR fluorescence in CD45.2⁺ cells over CD45.1⁺ cells within each animal. Each symbol represents an individual mouse. (G) Quantification of MitoTracker Green fluorescence in Lin^–cKit⁺Sca1⁺CD48^–CD150⁺PROCR⁺ cells isolated at 20 weeks after transplant, expressed as a ratio of the signal intensity in CD45.2⁺ cells over CD45.1⁺ cells within each animal. Each symbol represents an individual mouse. In all panels, mean and SEM are shown. *P < 0.05; **P < 0.01; ***P < 0.001; assessed by 1-way ANOVA (B, F, and G) or 2-way ANOVA (A and D) with Tukey’s multiple-comparison test.