Figure 3.
Loss of Bclaf1 does not alter HSC homing, survival, proliferation, or differentiation. (A) To assess BM homing, 30 000 HSPCs from adult CD45.2+Bclaf1f/f or Vav-Cre:Bclaf1f/f BM were sorted and transplanted into lethally irradiated CD45.1+ recipients. At 16 hours after transplant, percentage of live, Lin-CD45.2+ cells in the BM was quantified by flow cytometry (Bclaf1f/f, n = 5; Vav-Cre:Bclaf1f/f, n = 5). (B) Cell death assessed by percentage of annexin V–positive HSCs in E15.5 fetal liver (Bclaf1f/f, n = 4; Vav-Cre:Bclaf1f/f, n = 4), E17.5 fetal liver (Bclaf1f/f, n = 4; Vav-Cre:Bclaf1f/f, n = 6), and adult BM (Bclaf1f/f, n = 7; Vav-Cre:Bclaf1f/f, n = 7). (C) Proliferation assessed by in vivo BrdU incorporation in HSCs of E15.5 fetal liver (Bclaf1f/f, n = 4; Vav-Cre:Bclaf1f/f, n = 5), E17.5 fetal liver (Bclaf1f/f, n = 4; Vav-Cre:Bclaf1f/f, n = 5), and adult BM (Bclaf1f/f, n = 7; Vav-Cre:Bclaf1f/f, n = 7). (D-E) Myeloid potential was assessed by culturing sorted HSCs from adult Bclaf1f/f or Vav-Cre:Bclaf1f/f BM in myeloid differentiation media for 6.5 days; cells were then stained for indicated myeloid cell markers (D) and total numbers were enumerated (Bclaf1f/f, n = 7; Vav-Cre:Bclaf1f/f, n = 5) (E). (F-G) Bclaf1f/f or Vav-Cre:Bclaf1f/f E17.5 fetal HSCs were sorted and cultured in complete MethoCult media. Total CFUs (F) and number of CFU with each classification (G) were quantitated at day 10 of culture (Bclaf1f/f, n = 18; Vav-Cre:Bclaf1f/f, n = 18). (H-L) A total of 150 sorted HSCs from donor CD45.2+Bclaf1f/f or Vav-Cre:Bclaf1f/f fetal livers were transplanted with 300 000 CD45.1+ wild-type recipient BM cells into lethally irradiated CD45.1+ recipient mice. All analyses were conducted at 8 weeks after transplant (Bclaf1f/f, n = 12; Vav-Cre:Bclaf1f/f, n = 10). (H) Schematic of 150 HSC competitive transplant. (I) Total BM counts. (J) Frequency of CD45.2+ donor-derived HSCs. (K) Percentage of CD45.2+ donor-derived cells within each HSPC population. BM cells were gated on each HSPC population then on CD45.2. (L) HSPC populations as percentage of donor CD45.2+ BM cells. BM cells were gated on CD45.2+, then on each HSPC group within that donor population. Data in all panels are mean ± SD and statistical significance was determined by unpaired 2-tailed Student t test. ∗∗P ≤ .01; ∗∗∗∗P ≤ .0001. CFU, colony-forming unit; CFU-E, colony-forming unit–erythroid; CFU-G, colony-forming unit–granulocyte; CFU-GEMM, colony-forming unit–granulocyte-erythroid-macrophage-megakaryocyte; CFU-GM, colony-forming unit–granulocyte-macrophage; CFU-M, colony-forming unit–macrophage; IR, irradiation; ns, not significant; ST, short-term.

Loss of Bclaf1 does not alter HSC homing, survival, proliferation, or differentiation. (A) To assess BM homing, 30 000 HSPCs from adult CD45.2+Bclaf1f/f or Vav-Cre:Bclaf1f/f BM were sorted and transplanted into lethally irradiated CD45.1+ recipients. At 16 hours after transplant, percentage of live, Lin-CD45.2+ cells in the BM was quantified by flow cytometry (Bclaf1f/f, n = 5; Vav-Cre:Bclaf1f/f, n = 5). (B) Cell death assessed by percentage of annexin V–positive HSCs in E15.5 fetal liver (Bclaf1f/f, n = 4; Vav-Cre:Bclaf1f/f, n = 4), E17.5 fetal liver (Bclaf1f/f, n = 4; Vav-Cre:Bclaf1f/f, n = 6), and adult BM (Bclaf1f/f, n = 7; Vav-Cre:Bclaf1f/f, n = 7). (C) Proliferation assessed by in vivo BrdU incorporation in HSCs of E15.5 fetal liver (Bclaf1f/f, n = 4; Vav-Cre:Bclaf1f/f, n = 5), E17.5 fetal liver (Bclaf1f/f, n = 4; Vav-Cre:Bclaf1f/f, n = 5), and adult BM (Bclaf1f/f, n = 7; Vav-Cre:Bclaf1f/f, n = 7). (D-E) Myeloid potential was assessed by culturing sorted HSCs from adult Bclaf1f/f or Vav-Cre:Bclaf1f/f BM in myeloid differentiation media for 6.5 days; cells were then stained for indicated myeloid cell markers (D) and total numbers were enumerated (Bclaf1f/f, n = 7; Vav-Cre:Bclaf1f/f, n = 5) (E). (F-G) Bclaf1f/f or Vav-Cre:Bclaf1f/f E17.5 fetal HSCs were sorted and cultured in complete MethoCult media. Total CFUs (F) and number of CFU with each classification (G) were quantitated at day 10 of culture (Bclaf1f/f, n = 18; Vav-Cre:Bclaf1f/f, n = 18). (H-L) A total of 150 sorted HSCs from donor CD45.2+Bclaf1f/f or Vav-Cre:Bclaf1f/f fetal livers were transplanted with 300 000 CD45.1+ wild-type recipient BM cells into lethally irradiated CD45.1+ recipient mice. All analyses were conducted at 8 weeks after transplant (Bclaf1f/f, n = 12; Vav-Cre:Bclaf1f/f, n = 10). (H) Schematic of 150 HSC competitive transplant. (I) Total BM counts. (J) Frequency of CD45.2+ donor-derived HSCs. (K) Percentage of CD45.2+ donor-derived cells within each HSPC population. BM cells were gated on each HSPC population then on CD45.2. (L) HSPC populations as percentage of donor CD45.2+ BM cells. BM cells were gated on CD45.2+, then on each HSPC group within that donor population. Data in all panels are mean ± SD and statistical significance was determined by unpaired 2-tailed Student t test. ∗∗P ≤ .01; ∗∗∗∗P ≤ .0001. CFU, colony-forming unit; CFU-E, colony-forming unit–erythroid; CFU-G, colony-forming unit–granulocyte; CFU-GEMM, colony-forming unit–granulocyte-erythroid-macrophage-megakaryocyte; CFU-GM, colony-forming unit–granulocyte-macrophage; CFU-M, colony-forming unit–macrophage; IR, irradiation; ns, not significant; ST, short-term.

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