Efficient deletion of Atg5 in CD207+ DC subsets affects Langerhans cells but not cDC1. (A) Representative electrophoresis of genotyping PCR. Left panel: floxed, wild-type (WT) and exon 3-deleted (KO) alleles of Atg5. Right panel: wild-type and Cre knock-in alleles of Cd207. (B)Atg5 mRNA expression in sorted epidermal CD45⁺ MHCII+ TCRγδ⁻ LCs from control (Atg5^WT and Atg5^WT/Δ) and Atg5^ΔCd207 mice. Fold changes were calculated relative to mRNA expression in LCs of Atg5^WT control mice. Data are pooled from at least three independent experiments, with each point corresponding to one individual mouse. Statistical analysis: Kruskal–Wallis one-way ANOVA followed by Dunn’s multiple comparison test (*, P < 0.05; ns, P > 0.05). (C) Gating strategy used to sort lymph nodes MHCII⁺ CD207− FSA high dermal DCs (dDCs), MHCII⁺ CD207⁺ CD103⁻ LCs (LCs) and MHCII⁺ CD207⁺ CD103⁺ cDC1 (CD103⁺). Red dots in the top panel depict the backgating of CD207⁺ LCs/cDC1. (D)Atg5 mRNA expression in LCs, cDC1, and CD207− dDCs sorted from pooled lymph node cell suspensions of at least three control (Atg5^WT and Atg5^WT/Δ) or Atg5^ΔCd207 mice. Fold changes were calculated relative to mRNA expression in cells of Atg5^WT control mice. ND, not detectable. (E) Representative dot plots for the identification of CD207⁺ CD11b⁺ LCs, CD207⁺ CD11b⁻ cDC1, CD207⁻ CD11b⁺ cDC2/macrophages and CD207⁻ CD11b⁻ (DN, double-negative) DCs among live CD45⁺ lineage- CD11c⁺ MHCII⁺ skin DCs in whole skin cell suspensions from Atg5^WT and Atg5^ΔCd207 mice (lineage markers: B220, NK1.1, Ly6G and CD3). (F) Percentages of LCs and cDC1 among skin DCs. Data are pooled from at least three independent experiments, with each point corresponding to one individual mouse. Statistical analysis: Kruskal–Wallis one-way ANOVA followed by Dunn’s multiple comparison test (*, P < 0.05; ns, P > 0.05).