Figure 2. Deletion of Raptor impairs granulocyte and B cell development but does not alter progenitor cell survival or proliferation.

(A) Flow cytometric analysis of BM-MNCs from control and Raptor-deficient mice. Images represent data for HSCs/MPPs (K⁺S⁺L^–), CMPs (Lin^–c-Kit⁺Sca-1^–FcgRIII/II^–CD34⁺), GMPs (Lin^–c-Kit⁺Sca-1^–FcgRIII/II⁺CD34⁺), MEPs (Lin-c-Kit⁺Sca-1^–FcgRIII/II^–CD34^–), CLPs (IL-7Rα⁺Lin-c-Kit^midSca-1^mid), myeloid lineage cells (Mac-1/Gr-1), and B lineage cells (B220/IgM). Values are the mean percentage ± SD of the specified subpopulation among total BM-MNCs (n = 4). (B) Apoptosis. BM-MNCs from control and Raptor-deficient mice were subjected to TUNEL staining, and the indicated subpopulations were analyzed by flow cytometry. Data shown are the mean ± SD of TUNEL⁺ cells (n = 3). (C) Cell cycle. BrdU was injected i.p. into mice 2 hours prior to sacrifice. The indicated BM cell populations were stained with an anti-BrdU Ab and analyzed by flow cytometry. Data are the mean percentage ± SD of BrdU⁺ cells (n = 3). (D and E) Colony-forming ability. K⁺S⁺L^– cells (D) and GMP cells (E) were isolated from BM-MNCs of control and Raptor-deficient mice and cultured for 10 days in semisolid medium. Data are the mean colony number ± SD (n = 3). Colony diameters are indicated. (F) Flow cytometric analysis of the differentiation into myeloid and B lineage cells of control and Raptor-deficient K⁺S⁺L^– cells cultured on stromal cells. Raptor^fl/fl or Raptor^fl/flCreER K⁺S⁺L^– cells were cultured on a layer of OP-9 stromal cells for 2 weeks in the presence or absence of TAM. Data shown are the percentage ± SD of B220⁺ Mac-1⁺ cells among CD45⁺-gated cells (n = 3). One flow cytometric analysis representative of 3 independent experiments is shown. *P < 0.05, **P < 0.01 (Student’s t test).