mTOR deletion decreased GMPs and inhibits the ability to differentiate into monocytes/macrophages. (A) Myeloid progenitor subpopulations of SCID mice with or without RPM treatment were detected by flow cytometry. Plots shown were the gated Lin⁻sca-1⁻c-kit⁺ cells. The percentages (B) and the absolute cell numbers (C) of myeloid progenitor cell populations in the bone marrow of control and RPM-treated mice were summarized (mean ± SD; n = 6 mice each group). (D) BrdU incorporation in bone marrow-derived GMPs was detected by flow cytometry 24 hours after injection of BrdU. The percentages (E) and the cell numbers (F) of BrdU⁺ GMPs were shown. Data are representative of 3 independent experiments. (G) GMPs of control and RPM-treated SCID bone marrow were stained with anti-annexin-V and propidium iodide by flow cytometry analysis. The percentage (H) and cell number (I) of the alive cells were shown (mean ± SD; n = 3 mice). (J) A total of 5 × 10⁴ GMPs purified from control and RPM-treated mice and were seeded in the methylcellulose supplemented with IL-3, IL-6, SCF, and M-CSF. The assays were performed in triplicate and were photographed as the pictures presented. The colonies (K) and the macrophage cell number per colony (L) were calculated. Data are representative of 3 independent experiments. (M) GMPs sorted from tamoxifen-treated WT and ER-mTOR KO mice were cultured in the presence of M-CSF for 5 days in vitro and stained with anti-CD11b mAb and anti-F4/80 mAb. Then the phenotypes of the samples were analyzed by flow cytometry, as indicated. The decreased percentages (N) and the cell numbers (O) of CD11b⁺F4/80⁺ macrophages in mTOR-deficient GMPs were shown. Data are representative of 3 independent experiments. Data were expressed as mean ± SD. Three independent experiments with similar results were performed. *P < .05; **P < .01; ***P < .001 (WT vs ER-mTOR).