Gating strategy, quantification of deletion of Mpc2, and Mpc2 retroviral rescue of MPC2-deficient myeloid cells. (A) Representative flow cytometry gating strategies for splenic Ly6C^hi and Ly6C^lo monocytes and neutrophils. (B) Schematic representation of Mpc2 retroviral bone marrow chimera experiments to assess ability to rescue Mpc2 KO (+tam) or control (no tam) Ly6C^hi monocytes and neutrophils. C-kit⁺ cells were enriched from the bone marrow of Mpc2^fl/fl; ROSA26 CreER^+/− mice and transduced with Mpc2 retrovirus prior to transplantation alongside bone marrow from WT mice into irradiated recipients. (C) Representative flow cytometry plots of Mpc2 retrovirus (RV) expression in HSCs, Ly6C^hi monocytes, and neutrophils of an individual control or KO mouse. (D)Mpc2 transduction in HSCs paired with Ly6C^hi monocytes (top) or neutrophils (bottom) from the same mouse (n = 6 for untreated and n = 4 for tamoxifen). Data are pooled from two independent experiments. *, P < 0.05; **, P < 0.01 by Student’s two-tailed paired t test. (E) Quantitative PCR analysis of genomic Mpc2 deletion in Mpc2 chimeras. CD45.2⁺ cells from the bone marrow or spleen were sorted for genomic DNA extraction. DNA quantification within loxP sites was quantified relative to WT cells and GAPDH. Mean values ± SEM are shown. Data are pooled from two independent experiments. ****, P < 0.0001 by one-way ANOVA with post-hoc Tukey’s multiple comparisons test. (F) Raw chimerism values for Mpc2 chimeras shown in Fig. 2 E. Each symbol represents an individual mouse. Mean values ± SEM are shown, and data are pooled from three independent experiments. ****, P < 0.0001 by two-way ANOVA with post-hoc Tukey’s multiple comparisons test. (G) Representative flow cytometry gating strategies for CDPs and CMoPs in the bone marrow.