Effects of mTOR inhibition in primary progenitors and in vivo. (A) Surface expression of Sca-1 and c-Kit in mouse bone marrow progenitors (mBM) assessed by flow cytometry. c-Kit–positive and Sca-1–negative cells were sorted and used for myeloid differentiation. (B) Relative percentage of CD11b and Gr-1–positive cells in mouse bone marrow progenitors with or without rapamycin treatment (100nM), measured with flow cytometry. Cells were induced to differentiate in the presence of G-CSF (100 ng/mL, 10 days). Four separate experiments were conducted, and quantification of 3 replicates of a typical experiment are shown. Each bar represents the mean ± SEM (error bars). All values were normalized to the level (= 1) in cells without rapamycin treatment. *P < .05. (C) Relative percentage of CD11b and Gr-1–positive cells in mouse bone marrow progenitors with or without mTOR, Raptor, and S6K1 depletion, measured with flow cytometry. mTORshRNA1, Raptor shRNA1, and S6K1 shRNA were used to deplete mTOR, Raptor, and S6K1, respectively. NT (nontargeting) denotes cells infected with virus containing the empty retrovector (latent membrane protein or pMKO). Each bar represents the mean ± SEM (error bars) of 4 separate experiments. All values were normalized to the level (= 1) in cells without rapamycin treatment. *P < .05. (D) Relative percentage of lymphocytes in peripheral blood after 1-week rapamycin administration in vivo. “Control” and “Rap” indicate injection of vehicle and rapamycin, respectively. (E) Relative percentage of neutrophils and GMPs in the bone marrow after 1-week rapamycin administration in vivo. “Control” and “Rap” indicate injection of vehicle and rapamycin, respectively. (D-E) Three separate experiments were conducted, and in each experiment 10 mice were used. Each bar represents the mean ± SEM (error bars). All values were normalized to the level (= 100%) in cells without rapamycin treatment. *P < .05.