Figure 2
Figure 2. Loss of p53 rescues Mysm1−/− HSPC numbers and functions. (A) Flow cytometry plots gated on live KLS bone marrow cells and showing the expression of Flt3 and CD34. Stem and progenitor cell gates and population frequencies of representative samples from each group are shown. (B) Lin−cKit+Sca1+CD34−Flt3− LT-HSCs, Lin−cKit+Sca1+CD34+Flt3− ST-HSCs, and Lin−cKit+Sca1+CD34+Flt3+ MPPs as a percentage of KLS cells in the bone marrow of the mice. All data are from 4 to 5 mice per group, and are representative of 2 independent experiments. Absolute cell numbers are provided in supplemental Figure 4. (C-H) Competitive bone marrow transplantations, with the total bone marrow from Mysm1−/−p53+/+, Mysm1−/−p53+/−, or Mysm1−/−p53−/− mice mixed in a 1:1 ratio with CD45.1-marked wild-type bone marrow and injected into separate groups of lethally irradiated recipients. The recipient mice were analyzed for the relative contribution of Mysm1-deficient (CD45.2) and wild-type (CD45.1) cells to hematopoiesis at 24 weeks after the reconstitution. (C) Flow cytometry histograms of the bone marrow (top panel) and spleen (middle and bottom panels) of the chimeras gated on CD11b+ myeloid lineage cells, B220+ B cells, and CD4+ T cells, respectively. Histogram gates indicate CD45.1− (Mysm1−/−) and CD45.1+ (wild-type) donor cells. (D-E) Percentage contribution of Mysm1−/−p53+/+, Mysm1−/−p53+/−, or Mysm1−/−p53−/− cells to (D) myeloid lineage cells (CD11b+) in the recipient bone marrow, B-lineage cells (B220+) in the recipient bone marrow and spleen, and (E) total thymocytes and splenic CD4 and CD8 T cells. (F) Flow cytometry histograms of the bone marrow of the chimeras gated on LT-HSCs (Lin−cKit+Sca1+CD34−Flt3−), ST-HSCs (Lin−cKit+Sca1+CD34+Flt3−), and MPPs (Lin−cKit+Sca1+CD34+Flt3+). Histogram gates indicate CD45.1− (Mysm1−/−) and CD45.1+ (wild-type) donor cells. (G-H) Percentage contribution of Mysm1−/−p53+/+, Mysm1−/−p53+/−, or Mysm1−/−p53−/− cells to the stem cell and progenitor cell populations in the recipients’ bone marrow, gated based on (G) KLS, CD34, Flt3 or (H) KLS, CD150, CD48 expression. Bars show means ± SEM; *P < .05, **P < .01, ***P < .001 using analysis of variance with the Bonferroni post-hoc test. KLS, Lin−cKit+Sca1+; LT-HSC, long-term HSC; NS, nonsignificant; SEM, standard error of the mean; ST-HSC, short-term HSC.

Loss of p53 rescues Mysm1−/− HSPC numbers and functions. (A) Flow cytometry plots gated on live KLS bone marrow cells and showing the expression of Flt3 and CD34. Stem and progenitor cell gates and population frequencies of representative samples from each group are shown. (B) Lin−cKit+Sca1+CD34−Flt3− LT-HSCs, Lin−cKit+Sca1+CD34+Flt3− ST-HSCs, and Lin−cKit+Sca1+CD34+Flt3+ MPPs as a percentage of KLS cells in the bone marrow of the mice. All data are from 4 to 5 mice per group, and are representative of 2 independent experiments. Absolute cell numbers are provided in supplemental Figure 4. (C-H) Competitive bone marrow transplantations, with the total bone marrow from Mysm1−/−p53+/+, Mysm1−/−p53+/−, or Mysm1−/−p53−/− mice mixed in a 1:1 ratio with CD45.1-marked wild-type bone marrow and injected into separate groups of lethally irradiated recipients. The recipient mice were analyzed for the relative contribution of Mysm1-deficient (CD45.2) and wild-type (CD45.1) cells to hematopoiesis at 24 weeks after the reconstitution. (C) Flow cytometry histograms of the bone marrow (top panel) and spleen (middle and bottom panels) of the chimeras gated on CD11b+ myeloid lineage cells, B220+ B cells, and CD4+ T cells, respectively. Histogram gates indicate CD45.1− (Mysm1−/−) and CD45.1+ (wild-type) donor cells. (D-E) Percentage contribution of Mysm1−/−p53+/+, Mysm1−/−p53+/−, or Mysm1−/−p53−/− cells to (D) myeloid lineage cells (CD11b+) in the recipient bone marrow, B-lineage cells (B220+) in the recipient bone marrow and spleen, and (E) total thymocytes and splenic CD4 and CD8 T cells. (F) Flow cytometry histograms of the bone marrow of the chimeras gated on LT-HSCs (Lin−cKit+Sca1+CD34−Flt3−), ST-HSCs (Lin−cKit+Sca1+CD34+Flt3−), and MPPs (Lin−cKit+Sca1+CD34+Flt3+). Histogram gates indicate CD45.1− (Mysm1−/−) and CD45.1+ (wild-type) donor cells. (G-H) Percentage contribution of Mysm1−/−p53+/+, Mysm1−/−p53+/−, or Mysm1−/−p53−/− cells to the stem cell and progenitor cell populations in the recipients’ bone marrow, gated based on (G) KLS, CD34, Flt3 or (H) KLS, CD150, CD48 expression. Bars show means ± SEM; *P < .05, **P < .01, ***P < .001 using analysis of variance with the Bonferroni post-hoc test. KLS, Lin−cKit+Sca1+; LT-HSC, long-term HSC; NS, nonsignificant; SEM, standard error of the mean; ST-HSC, short-term HSC.

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