Impaired lipid metabolism in ATG5-deficient Langerhans cells. (A–E) Flow cytometry quantification of mean intensity of fluorescence for (A) Phosphorylated AMPK, (B) GLUT1, (C) 2-NDBG uptake, (D) CD36, and (E) Bodipy C16 uptake in epidermal LCs obtained from 3-wk-old control (Atg5^WT and Atg5^WT/Δ) and Atg5^ΔCd207 mice. (F) Epidermal LCs sorted from Atg5^WT or Atg5^ΔCd207 mice and BMDCs derived from C57BL/6 mice were sequentially exposed to Oligomycin (OM), Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), rotenone (ROT) and antimycin A (AA), and oxygen consumption rates (OCR) were measured by a Seahorse XF96 analyzer throughout the experiment. Data are from one representative experiment out of three. (G) ATP production (OCR_baseline–OCR_OM), maximum respiration (Max; OCR_FCCP–OCR_AA+ROT), and spare respiratory capacity (SRC; OCR_FCCP–OCR_baseline) were calculated from the OCR curves. (H) Differentially expressed transcripts related to mitochondria (GO: 0005739) in Atg5^WT versus Atg5^ΔCd207 LCs. (I) Mitochondrial load for epidermal LCs of Atg5^WT, Atg5^WT/Δ, and Atg5^ΔCd207 mice, as measured by MFI of Mitotracker Green staining. (J) Representative dot plot of Mitotracker Green and Deep-Red staining and comparison of Mitotracker Deep-Red MFI of epidermal LCs obtained from control (Atg5^WT and Atg5^WT/Δ) and Atg5^ΔCd207 mice. (K) Representative dot plot of Mitosox Red staining and comparison of Mitosox^high percentage of epidermal LCs obtained from control (Atg5^WT and Atg5^WT/Δ) and Atg5^ΔCd207 mice. All data are pooled from at least three 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 (except g: two-way ANOVA followed by Šídák’s multiple comparisons test). (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, P > 0.05).