mTOR controls monocyte/macrophage development through STAT5/IRF8. (A) GMPs sorted from tamoxifen-treated WT and ER-mTOR KO mice were lysed and analyzed for the transcriptional factor expression of IRF8, nuclear receptor subfamily 4 group A member 1, CCR2, MAF BZIP transcription factor B, PU.1, runt-related transcription factor 1, SP1, CCAAT-enhancer-binding protein β, and C/EBPα. Three mice in each group were assayed. (B) GMPs sorted from tamoxifen-treated WT and ER-mTOR KO mice were cultured in the presence of IL-3+IL-6 and collected at day 0, day 1, and day 3. Then the samples were lysed and analyzed for the translational expression of CSF1R, IRF8, PU.1, C/EBβ LAP, C/EBPα, and mTOR by immunoblotting analysis. (C) GMPs sorted from tamoxifen-treated WT and ER-mTOR KO mice were stimulated with IL-3+IL-6 at the indicated points. Then the samples were lysed and analyzed by immunoblotting for phosphorylation of STAT3, STAT5, and S6. (D) GMPs were sorted from tamoxifen-untreated WT and ER-mTOR KO mice and cultured with 4-OHT in the presence and absence of STAT5 inhibitor, pimozide. The percentage (D) and cell number (E) of CD11b⁺CD115⁺ cells were calculated. The percentage (F) and cell number (G) of CD11b⁺F4/80⁺ macrophages were also detected. (H) 5 × 10⁴ sorted from tamoxifen-treated WT and ER-mTOR KO mice were seeded in the methylcellulose supplemented with IL-3, IL-6, SCF, M-CSF, and 4-OHT in the presence and absence of pimozide (5 µM). The experiment was performed in triplicate and was photographed as the pictures presented. Scale bars, 100 µm. The generated colonies (I) and the macrophage cell numbers per colony (J) were calculated. (K) GMPs sorted from WT and ER-mTOR mice were cultured in the presence of 4-OHT and pimozide at the indicated concentrations and analyzed for the transcriptional factor expression of IRF8. Data are shown as mean ± SD (n = 3). **P < .01; ***P < .001 compared with WT group; ***P < .001 compared with WT mice.