Journal: The Journal of Cell Biology

Article Title: Epidermal maintenance of Langerhans cells relies on autophagy-regulated lipid metabolism

doi: 10.1083/jcb.202403178

Figure Lengend Snippet: 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).

Article Snippet: Bodipy FL C16 , Bodipy 505/512 , N/A , Invitrogen , D3821.

Techniques: Flow Cytometry, Control, Comparison, MANN-WHITNEY, Staining, Marker

Journal: The Journal of Cell Biology

Article Title: Epidermal maintenance of Langerhans cells relies on autophagy-regulated lipid metabolism

doi: 10.1083/jcb.202403178

Figure Lengend Snippet: Lipid droplets of Atg5 WT Langerhans cells. Representative immunofluorescent staining of neutral lipids using the Bodipy dye on epidermal LCs of 3-wk-old Atg5 WT mice. CD207: green; Bodipy: red; DAPI: blue. Scale bar: 10 µm.

Article Snippet: Bodipy FL C16 , Bodipy 505/512 , N/A , Invitrogen , D3821.

Techniques: Staining

Journal: The Journal of Cell Biology

Article Title: Epidermal maintenance of Langerhans cells relies on autophagy-regulated lipid metabolism

doi: 10.1083/jcb.202403178

Figure Lengend Snippet: Lipid droplets of Atg5 ΔCd207 Langerhans cells. Representative immunofluorescent staining of neutral lipids using the Bodipy dye on epidermal LCs of 3-wk-old Atg5 ΔCd207 mice. CD207: green; Bodipy: red; DAPI: blue. Scale bar: 10 µm.

Article Snippet: Bodipy FL C16 , Bodipy 505/512 , N/A , Invitrogen , D3821.

Techniques: Staining

Journal: The Journal of Cell Biology

Article Title: Epidermal maintenance of Langerhans cells relies on autophagy-regulated lipid metabolism

doi: 10.1083/jcb.202403178

Figure Lengend Snippet: 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).

Article Snippet: Bodipy FL C16 , Bodipy 505/512 , N/A , Invitrogen , D3821.

Techniques: Flow Cytometry, Fluorescence, Control, Derivative Assay, Staining, Comparison

Journal: The Journal of Cell Biology

Article Title: Epidermal maintenance of Langerhans cells relies on autophagy-regulated lipid metabolism

doi: 10.1083/jcb.202403178

Figure Lengend Snippet: Autophagy-deficient Langerhans cells undergo ferroptosis. (A) Differentially expressed transcripts included in the KEGG ferroptosis pathway (mmu04216) in Atg5 WT versus Atg5 ΔCd207 LCs. No threshold was applied to fold changes. (B) MFI of CD71 at the surface of LCs. (C) MFI of FerroOrange staining, reflecting the concentration of ferrous iron (Fe 2+ ) in LCs. Data are pooled from five independent experiments, with each point representing one individual 3-wk-old mouse. (D) Quantification of lipid peroxidation for epidermal LCs of control ( Atg5 WT and Atg5 WT/Δ ) and Atg5 ΔCd207 mice, as measured by MFI of Bodipy-C11. Data are pooled from 10 independent experiments, with each point representing one individual 3-wk-old mouse. (E) Lipid peroxidation of Atg5 WT and Atg5 ΔCd207 LCs after overnight incubation with 50 µM ferrostatin-1. Data are pooled from four independent experiments, with each point representing one individual 3-wk-old mouse. Statistical analyses: (B–D) Kruskal–Wallis one-way ANOVA followed by Dunn’s multiple comparison test (***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, P > 0.05). (E) Two-way ANOVA followed by Sidak’s multiple comparison tests. (*, P < 0.05; ns, P > 0.05).

Article Snippet: Bodipy FL C16 , Bodipy 505/512 , N/A , Invitrogen , D3821.

Techniques: Staining, Concentration Assay, Control, Incubation, Comparison

Journal: The Journal of Cell Biology

Article Title: Epidermal maintenance of Langerhans cells relies on autophagy-regulated lipid metabolism

doi: 10.1083/jcb.202403178

Figure Lengend Snippet: Antibodies and reagents for flow cytometry and immunofluorescence microscopy

Article Snippet: Bodipy FL C16 , Bodipy 505/512 , N/A , Invitrogen , D3821.

Techniques: Flow Cytometry, Immunofluorescence, Luminex, SYBR Green Assay