Figure 2
Figure 2. Necdin is dispensable for steady-state hematopoiesis. (A) Normal BM cellularity in Ndn+m/−p mice. BM cells were isolated from the femurs and tibias of both hind legs. Absolute numbers of BM cells obtained from either Ndn+/+ or Ndn+m/−p mice were determined with a hemocytometer. Data shown are the mean number ± SD (n = 11). (B) Ndn-deficient mice exhibit normal myeloid, T-cell, and B-cell differentiation capacities. BM cells isolated from either Ndn+/+ or Ndn+m/−p mice were stained with anti-Mac1 and Gr-1 antibodies, anti-CD3 antibody, or anti-B220 antibody. The frequency of myeloid cells (Mac-1+/Gr-1+), T cells (CD3+), or B cells (B220+) in BM was determined by flow cytometry. Data shown are the mean number ± SD (n = 4). (C) Normal in vitro colony-forming capacity of Ndn+m/−p BM cells. BM cells from either Ndn+/+ or Ndn+m/−p mice were assessed by an in vitro colony formation assay (“Hematopoietic progenitor assays in vitro and in vivo”). Data are shown as the mean number ± SD of colonies (n = 4). (D) Colony-forming units in spleen at day 12 after transplantation (CFU-S12) are comparable between Ndn+/+ and Ndn+m/−p BM cells. Data are shown as mean colony number ± SD. No significant difference was observed between Ndn+/+ and Ndn+m/−p mice (Ndn+/+: n = 8, Ndn+m/−p: n = 5). (E) The frequency of apoptotic cells in CD34−/low KSL cells after cytokine depletion. FACS-purified CD34−/low KSL cells from Ndn+/+ and Ndn+m/−p mice were cultured without cytokines in serum-containing medium for the indicated times and stained with annexin V and 7-AAD. Data shown are the mean percentage ± SD of annexin V+ 7-AAD− cells (n = 3). No significant differences were detected between Ndn+/+ and Ndn+m/−p mice. (F) Apoptosis rates in BM cells after γ-irradiation. BM cells from Ndn+/+ or Ndn+m/−p mice harvested 12 hours after 6.5 Gy irradiation were evaluated for apoptosis using annexin V staining. Data are shown as the mean percentage ± SD of annexin V+ 7-AAD− CD34−/low KSL cells (n = 3). No significant differences were seen between Ndn+/+ and Ndn+m/−p mice.

Necdin is dispensable for steady-state hematopoiesis. (A) Normal BM cellularity in Ndn+m/−p mice. BM cells were isolated from the femurs and tibias of both hind legs. Absolute numbers of BM cells obtained from either Ndn+/+ or Ndn+m/−p mice were determined with a hemocytometer. Data shown are the mean number ± SD (n = 11). (B) Ndn-deficient mice exhibit normal myeloid, T-cell, and B-cell differentiation capacities. BM cells isolated from either Ndn+/+ or Ndn+m/−p mice were stained with anti-Mac1 and Gr-1 antibodies, anti-CD3 antibody, or anti-B220 antibody. The frequency of myeloid cells (Mac-1+/Gr-1+), T cells (CD3+), or B cells (B220+) in BM was determined by flow cytometry. Data shown are the mean number ± SD (n = 4). (C) Normal in vitro colony-forming capacity of Ndn+m/−p BM cells. BM cells from either Ndn+/+ or Ndn+m/−p mice were assessed by an in vitro colony formation assay (“Hematopoietic progenitor assays in vitro and in vivo”). Data are shown as the mean number ± SD of colonies (n = 4). (D) Colony-forming units in spleen at day 12 after transplantation (CFU-S12) are comparable between Ndn+/+ and Ndn+m/−p BM cells. Data are shown as mean colony number ± SD. No significant difference was observed between Ndn+/+ and Ndn+m/−p mice (Ndn+/+: n = 8, Ndn+m/−p: n = 5). (E) The frequency of apoptotic cells in CD34−/low KSL cells after cytokine depletion. FACS-purified CD34−/low KSL cells from Ndn+/+ and Ndn+m/−p mice were cultured without cytokines in serum-containing medium for the indicated times and stained with annexin V and 7-AAD. Data shown are the mean percentage ± SD of annexin V+ 7-AAD− cells (n = 3). No significant differences were detected between Ndn+/+ and Ndn+m/−p mice. (F) Apoptosis rates in BM cells after γ-irradiation. BM cells from Ndn+/+ or Ndn+m/−p mice harvested 12 hours after 6.5 Gy irradiation were evaluated for apoptosis using annexin V staining. Data are shown as the mean percentage ± SD of annexin V+ 7-AAD− CD34−/low KSL cells (n = 3). No significant differences were seen between Ndn+/+ and Ndn+m/−p mice.

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