Extended Data Figure 3. Mycl1 expression is restricted to dendritic cells in lymphoid and non-lymphoid tissues.

a, Shown are single-color histograms of GFP expression for the indicated cells derived from WT, Mycl1^+/gfp, and Mycl1^gfp/gfp BM cells cultured in FMS-like tyrosine kinase 3 ligand (Flt3L) for 10 days. cDCs (Flt3L cDC) were gated as CD11c⁺MHCII⁺SiglecH⁻ and pDCs (Flt3L pDC) were gated as CD11c^intSiglecH⁺. Numbers represent GFP⁺ cells as a percent of live cells. Data are representative of 3 independent experiments. b, Shown are two-color histograms of CD24 and SIRP-α expression for Flt3L cDCs as described in (a) from BM of WT mice (left panel) and Mycl1^+/gfp mice (middle and right panels). Flt3L cDCs from BM of Mycl1^+/gfp mice were pre-gated as either GFP⁺ or GFP⁻. Numbers represent the percent of cells in the indicated gate. c, Flt3⁺ CMPs purified by cell sorting from WT BM cells were cultured in Flt3L for 12 hours before transduction with control (Empty) retrovirus or with retrovirus expressing IRF8, L-Myc, or c-Myc. Cells were cultured for an additional 5 days and then stained for analysis. Shown is a two-color histogram of MHCII and SiglecH expression for each indicated transduction. cDCs are gated as MHCII⁺SiglecH⁻ and pDCs are gated as MHCII⁻SiglecH⁺. Numbers represent the percent of cells in the quadrant gate. d, Shown is a quantitation of undifferentiated (non-cDC or non-pDC) cells from (c). Data are from 4 independent transductions per retrovirus (bar, SD; n=4, one-way ANOVA Tukey's post hoc test). e, Cells from the spleen (left panel), inguinal lymph nodes (ILN, middle panel), and mesenteric lymph nodes (MLN, right panel) of WT, Myc^gfp/gfp, and Mycl1^+/gfp mice were stained for analysis. Shown are two-color histograms of CD11c and GFP expression for non-autofluorescent cells. Numbers represent percent of cells in the indicated gate. Data are representative of at least 5 independent experiments. f, Shown are single-color histograms of GFP expression for resident DCs (CD11c⁺MHCII^int, DC) and migratory DCs (CD11c^intMHCII⁺, mDC) from mesenteric lymph nodes (MLN, top panel) and inguinal lymph nodes (ILN, bottom panel) of WT (grey lines) and Mycl1^+/gfp mice (green lines). Resident DCs were further gated as CD24⁺SIRP-α⁻ (CD8α⁺ DC) and CD24⁻SIRP-α⁺ (CD8α⁻ DC). MLN mDCs were gated as CD103⁺CD11b⁻ (CD103⁺ mDC), CD103⁺CD11b⁺ (CD103⁺CD11b⁺ mDC), and CD103⁻CD11b⁺ (CD11b⁺ mDC). ILN mDCs were gated as CD103⁺CD11b⁻ (CD103⁺ mDC), CD103⁻CD11b⁺ (CD11b⁺ mDC), and CD103⁻CD11b^int/− (Langerhans cells). g, Cells from the brain, spleen, peritoneum, kidney, and liver of WT and Mycl1^+/gfp mice were stained for analysis. Shown are two-color histograms of F4/80 and GFP expression for microglia (CD45^intCD11b⁺), splenic red pulp macrophages (F4/80⁺autofluorescent^high, RPM), peritoneum macrophages (F4/80⁺CD11b⁺), liver and kidney macrophages (F4/80⁺CD11b^int), and liver DCs (CD11c⁺MHCII⁺). Live hematopoietic cells were pre-gated in all non-lymphoid tissues as CD45^+/int7AAD⁻. Numbers represent percent of cells in the indicated gate. Data are representative of 2-3 independent experiments(n=4 mice). *, p<0.01; ns, p>0.05.