Impaired autophagy increases the lipid storage compartments of Langerhans cells. (A) Differentially expressed transcripts related to lipid metabolism pathways in Atg5^WT versus Atg5^ΔCd207 LCs (GO:0046942, GO:0043269, GO:0017144, GO:0044272, GO:0051186, GO:0110096, GO:0046085, GO:1901615, GO:0015711, GO:0009166, and GO:0007584). (B) Flow cytometry quantification of the side scatter (SSC) MFI in epidermal LCs obtained from control (Atg5^WT and Atg5^WT/Δ) and Atg5^ΔCd207 mice. Data are pooled from 10 independent experiments, with each point representing one individual 3-wk-old mouse. Statistical analysis: Kruskal–Wallis one-way ANOVA followed by Dunn’s multiple comparison test (**, P < 0.01; ****, P < 0.0001; ns, P > 0.05). (C and D) Flow cytometry quantification of the Bodipy MFI in epidermal LCs obtained from C57BL/6 mice then treated with (C) etomoxir or (D) wortmannin. Data are pooled from at least three independent experiments, with each point corresponding to one individual 3-wk-old mouse. Statistical analysis: Mann–Whitney U test (*, P < 0.05). (*, P < 0.05; **, P < 0.01). (E) Flow cytometry quantification of Bodipy MFI in epidermal LCs obtained from control (Atg5^WT and Atg5^WT/Δ) and Atg5^ΔCd207 mice. Data are pooled from four independent experiments, with each point representing one individual 3-wk-old mouse. Statistical analysis: Kruskal–Wallis one-way ANOVA followed by Dunn’s multiple comparison test (**, P < 0.01; ns, P > 0.05). (F) Immunofluorescent staining of MHCII+ epidermal LCs obtained from Atg5^WT (upper panels) and Atg5^ΔCd207 (lower panels) mice and stained with Bodipy or anti-Perilipin-2 antibody. Scale bars: 10 µm. (G and H). Quantification of (G) Bodipy+ and (H) Perilipin-2+ vesicles in LCs obtained from control (Atg5^WT and Atg5^WT/Δ) and Atg5^ΔCd207 mice. Data are representative of three independent experiments and presented as violin plots (solid line, median; dashed lines, first and third quartiles; n > 100 cells from a total of three 3-wk-old mice). Statistical analysis: Kruskal–Wallis one-way ANOVA followed by Dunn’s multiple comparison test ***, P < 0.001; ****, P < 0.0001). (I) Representative half-set overlay of LysoTracker-Red (left panel), and LysoSensor (right panel) staining. (J) Comparison of the ratio of MFI of each marker for epidermal LCs of control (Atg5^WT and Atg5^WT/Δ) and Atg5^ΔCd207 mice. Data are pooled from at least three independent experiments, with each point corresponding to one 3-wk-old individual mouse. Statistical analysis: Kruskal–Wallis one-way ANOVA followed by Dunn’s multiple comparison test (ns, P > 0.05).