p53 mediates loss of hematopoietic stem cell function and lymphopenia in Mysm1 deficiency by Jad I. Belle, David Langlais, Jessica C. Petrov, Mercedes Pardo, Russell G. Jones, Philippe Gros, and Anastasia Nijnik Blood Volume 125(15):2344-2348 April 9, 2015 ©2015 by American Society of Hematology

p53 mediates the developmental abnormalities and impaired lymphocyte differentiation in the Mysm1−/− mice. p53 mediates the developmental abnormalities and impaired lymphocyte differentiation in the Mysm1−/− mice. Data from mice of the following genotypes is presented: Mysm1+/−p53+/+, Mysm1+/−p53−/−, Mysm1−/−p53−/−, and Mysm1−/−p53+/+. Mysm1+/− mice were shown to be phenotypically indistinguishable from wild-type throughout our previous studies, and in this case the comparison against Mysm1+/− allowed the experimental mice to be bred as age-matched littermates. (A) Representative photograph of the mice, showing that loss of p53 rescues the growth retardation, as well as the tail and hind-limb abnormalities of the Mysm1−/− mice. (B) Mouse lengths and weights. (C) Representative flow cytometry plots of mouse bone marrow, stained for B220 and CD19, and gated on live cells. Average percentage of cells within the B220+CD19+ B-cell lineage gate is shown. (D) Representative flow cytometry plots of the mouse spleen, stained for B220 and CD4, and gated on live cells. Average percentages of cells within the B220+ B-cell gate and CD4+ T-helper cell gate are shown. (E) Numbers of B- and T-lineage cells in the spleen of the mice; cells gated as B220+, CD4+, or CD8+. (F) Numbers of pro-B and pre-B cells (B220+IgM−IgD−), immature B cells (B220+IgM+IgD−), and mature B cells (B220+IgM+IgD+) in the bone marrow of the mice. (G) Numbers of double-negative (CD4−CD8−), double-positive (CD4+CD8+), and CD4 and CD8 single-positive thymocytes in the mice. Bars show means ± SEM; *P < .05, **P < .01, ***P < .001; all data are from 4 to 5 mice per group, and are representative of 3 independent experiments. NS, nonsignificant using analysis of variance with the Bonferroni post-hoc test; SEM, standard error of the mean. Jad I. Belle et al. Blood 2015;125:2344-2348 ©2015 by American Society of Hematology

Loss of p53 rescues Mysm1−/− HSPC numbers and functions. 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. Jad I. Belle et al. Blood 2015;125:2344-2348 ©2015 by American Society of Hematology