Figure 6

Assessing the cell-intrinsic role of MYSM1 DUB catalytic activity in hematopoiesis and leukocyte development with competitive bone marrow transplantation. (A) Schematic representation of the mouse-to-mouse competitive bone marrow transplantation study. Wild type CD45.1⁺ bone marrow cells were mixed in a 1:1 ratio with Cre^ERT2 transgenic bone marrow cells of Mysm1^fl/+, Mysm1^fl/fl, or Mysm1^fl/DN genotypes, and the mixes were transplanted into three independent cohorts of lethally irradiated wild type CD45.1⁺ recipient mice. Following full hematopoietic reconstitution, the chimeric mice were administered with tamoxifen to induce the Mysm1^fl to Mysm1^Δ allele conversion. Clipart images were used toward the preparation of the Figure (http://clipart-library.com). (B,C) The relative contribution of Mysm1^Δ/DN, Mysm1^Δ/Δ, and control Mysm1^Δ/+ cells to the different hematopoietic and immune cell populations was evaluated by flow cytometry, quantifying the proportion of CD45.2⁺CD45.1⁻ cells within each cell population. Data is from 3–5 mice per group; bars represent means ± SEM; statistical analysis uses ANOVA and Dunnett’s post-hoc test comparing each group to the Mysm1^Δ/+ control; *p < 0.05, **p < 0.01, ***p < 0.001, or NS—not significant. Data is presented for the following cell populations: (B) splenic B cells (CD19⁺CD3⁻), CD4 T cells (CD3⁺CD4⁺CD8⁻), CD8 T cells (CD3⁺CD4⁻CD8⁺), and NK cells (CD3⁻NK1.1⁺); bone marrow monocytes (CD11b⁺Ly6C^hiLy6G^lo), neutrophils (CD11b⁺Ly6C^loLy6G^hi), and erythroid cells (CD71⁺); (C) bone marrow stem and multipotent progenitors (LKS, Lin⁻cKit⁺Sca1⁺), common myeloid progenitors (CMP, Lin⁻cKit⁺Sca1⁻CD34⁺CD16/32⁻), granulocyte monocyte progenitors (GMP, Lin⁻cKit⁺Sca1⁻CD34⁺CD16/32⁺), common lymphoid progenitors (CLP, Lin⁻cKit^loSca1^loIL7Ra⁺CD16/32⁻), megakaryocyte erythroid progenitors (MEP, Lin⁻cKit⁺Sca1⁻CD34⁻CD16/32⁻), and megakaryocyte progenitors (MkP, Lin⁻cKit⁺Sca1⁻CD16/32⁻CD150⁺CD41⁺). (D) Representative flow cytometry plots showing the analyses of splenic B cells, splenic T cells, and bone marrow neutrophils for CD45.1 versus CD45.2 marker expression; percentage of cells in the gates is shown as mean ± st. dev. of all the mice in each group. Gates for CD45.1⁺ and CD45.2⁺ cells were set independently for each cell population using control non-chimeric WT-B6 (CD45.2) and WT-SJL (CD45.1) mice, as shown in Fig. S4E.