Figure 2. GMPs and MDPs produce both Ly6C^hi and Ly6C⁻ CD43⁺ monocytes.

(A) To identify monocyte subsets in the bone marrow, blood and spleen of wild-type mice, Ly6C and CD43 expression by CD11b⁺ CD115⁺ Ly6G⁻ F4/80⁻ cells was assessed by flow cytometry (see Figure S4A for gating strategy). (B–C) Ly6C^hi, Ly6C⁻ CD43⁺ and Ly6C⁻ CD43⁻ subsets were assessed in the bone marrow, blood and spleen of wild-type (B–C), Irf8-deficient (B) and Nur77 (Nr4a1)-deficient (C) mice with additional gating of CCR2⁺ cells to identify Ly6C^hi monocytes due to the presence of incompletely differentiated Ly6C^int monoblasts in Irf8-deficient mice (Figure S4C and (Yanez et al., 2015)). Data are presented as mean plus standard deviation of 5 mice, and statistical significance was assessed by Student’s t-test (*p<0.05, **p<0.01, ***p<0.001). (D–E) 25,000 GMPs, MDPs or MPs+cMoPs isolated from wild-type (D–E), Irf8-deficient (D) or Nur77 (Nr4a1)-deficient (E) donor mice (all CD45.2) were injected i.v. into congenic CD45.1 recipient mice (non-irradiated) on day 0. Spleens were harvested from recipient mice at the indicated timepoints after progenitor injection, and splenocytes were enriched for CD45.2⁺ (donor-derived) cells prior to assessment of donor-derived Ly6C^hi (and CCR2⁺), Ly6C⁻ CD43⁺ and Ly6C⁻ CD43⁻ cells by flow cytometry (see Figure S4D for gating strategy). Data are presented as mean plus standard error of 3–4 mice that received progenitors in independent experiments. (F) CD11c and Zbtb46 expression by Ly6C^hi, Ly6C⁻ CD43⁺ and Ly6C⁻ CD43⁻ cells in the bone marrow, blood and spleen of Zbtb46-GFP transgenic mice was assessed by flow cytometry.