Journal: Neural Regeneration Research

Article Title: NOX4 exacerbates Parkinson’s disease pathology by promoting neuronal ferroptosis and neuroinflammation

doi: 10.4103/NRR.NRR-D-23-01265

Figure Lengend Snippet: NOX4 inhibition mitigates MPP + -induced ferroptosis in MN9D cells. (A) Cell viability was measured by CCK-8 in MN9D cells with/without MPP + 0.5 mM, GLX351322 (selective NOX4 antagonist) 5 µM, and Fer-1 (a classic ferroptosis inhibitor) 1 µM treatment for 24 hours ( n = 5/group). (B) Cell viability was measured by CCK-8 in MN9D cells with/without erastin 5 µM, GLX351322 5 µM, and Fer-1 1 µM treatment for 24 hours ( n = 5/group). (C, D) Representative images of C11-BODIPY 581/591 fluorescence in different groups and quantification of the green/red fluorescent ratio ( n = 3/group). A higher green/red fluorescence ratio indicates more lipid peroxidation. (E) Representative TEM images of MN9D cells with/without MPP + and GLX351322 treatment. Yellow arrows indicate shrunken mitochondria. (F–H) Western blot analysis of ACSL4 and GPX4 expression in MN9D cells from different groups; bands were quantified by densitometric analysis ( n = 3/group). (I–L) 4-HNE, MDA, and SOD levels and the GSH/GSSG ratio were measured in MN9D cells from different groups ( n = 3/group). Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. One-way analysis of variance with Tukey’s multiple comparisons test was conducted. The experiments were repeated at least three times. Scale bars: 50 µm in C and 2 µm or 500 nm in E. 4-HNE: 4-Hydroxynonenal; ACSL4: acyl-CoA synthetase long-chain family member 4; Fer-1: ferrostatin-1; GLX: GLX351322 (a selective NOX4 antagonist); GPX4: glutathione peroxidase 4; GSH/GSSG: glutathione/glutathione disulfide; MDA: malondialdehyde; MPP + : 1-methy-4-phenylpyridinium; SOD: superoxide dismutase.

Article Snippet: MN9D cells were incubated with 1 µM C11-BODIPY 581/591 fluorescent probe (Thermo Fisher Scientific, D3861) for 30 minutes.

Techniques: Inhibition, CCK-8 Assay, Fluorescence, Western Blot, Expressing

Journal: Neural Regeneration Research

Article Title: NOX4 exacerbates Parkinson’s disease pathology by promoting neuronal ferroptosis and neuroinflammation

doi: 10.4103/NRR.NRR-D-23-01265

Figure Lengend Snippet: The NOX4-PKCα axis modulates MPP + /MPTP-induced ferroptosis in MN9D cells and mouse brains. (A, B) Western blot analysis of p-PKCα and PKCα in the substantia nigra of mice from different groups; bands were quantified by densitometric analysis ( n = 6/group). (C) Coimmunoprecipitation of PKCα from MN9D cells using an anti-NOX4 antibody. (D) Coimmunoprecipitation of PKCα from mouse brains using an anti-NOX4 antibody. (E) PLA assay of the association between NOX4 and PKCα in MN9D cells. A red PLA signal means that PKCα and NOX4 were located < 40 nm away from each other. (F) Cell viability was measured by cell counting kit-8 in MN9D cells with/without MPP + 0.5 mM and BIM 5 µM treatment for 24 hours ( n = 5/group). (G) Representative TEM images of MN9D cells with/without MPP + and BIM treatment. Yellow arrows indicate shrunken mitochondria. (H, I) Representative C11-BODIPY 581/591 images from different groups and quantification of the green/red fluorescent ratio ( n = 3/group). A higher green/red fluorescence ratio indicates more lipid peroxidation. (J–M) 4-HNE, MDA, and SOD levels and the GSH/GSSG ratio were measured in MN9D cells from different groups ( n = 3/group). Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. One-way analysis of variance with Tukey’s multiple comparisons test was conducted. Scale bars: 10 µm in E, 2 µm or 500 nm in G, 50 µm in H. 4-HNE: 4-Hydroxynonenal; BIM: bisindolylmaleimide I (protein kinase C inhibitor); Ctrl: control; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; GLX: GLX351322 (a selective NOX4 antagonist); GSH/GSSG: glutathione/glutathione disulfide; MDA: malondialdehyde; MPTP: 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine; NOX4: NADPH oxidase 4; SOD: superoxide dismutase.

Article Snippet: MN9D cells were incubated with 1 µM C11-BODIPY 581/591 fluorescent probe (Thermo Fisher Scientific, D3861) for 30 minutes.

Techniques: Western Blot, Cell Counting, Fluorescence, Control

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: 50,000 enriched CD11b+ LCs were seeded and incubated for 10 min at 37°C with 2 mM Bodipy-C11 (581/591) (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid; Invitrogen) in PBS.

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: 50,000 enriched CD11b+ LCs were seeded and incubated for 10 min at 37°C with 2 mM Bodipy-C11 (581/591) (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid; Invitrogen) in PBS.

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

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: Epidermal cells were then incubated with Mitotracker, Mitosox, ER-tracker, Bodipy, or Bodipy-C16 according to the manufacturer’s instructions (Invitrogen) before fixation using 2% PFA in PBS for 15 min at RT.

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: Epidermal cells were then incubated with Mitotracker, Mitosox, ER-tracker, Bodipy, or Bodipy-C16 according to the manufacturer’s instructions (Invitrogen) before fixation using 2% PFA in PBS for 15 min at RT.

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: Epidermal cells were then incubated with Mitotracker, Mitosox, ER-tracker, Bodipy, or Bodipy-C16 according to the manufacturer’s instructions (Invitrogen) before fixation using 2% PFA in PBS for 15 min at RT.

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: Epidermal cells were then incubated with Mitotracker, Mitosox, ER-tracker, Bodipy, or Bodipy-C16 according to the manufacturer’s instructions (Invitrogen) before fixation using 2% PFA in PBS for 15 min at RT.

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: Epidermal cells were then incubated with Mitotracker, Mitosox, ER-tracker, Bodipy, or Bodipy-C16 according to the manufacturer’s instructions (Invitrogen) before fixation using 2% PFA in PBS for 15 min at RT.

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: Epidermal cells were then incubated with Mitotracker, Mitosox, ER-tracker, Bodipy, or Bodipy-C16 according to the manufacturer’s instructions (Invitrogen) before fixation using 2% PFA in PBS for 15 min at RT.

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

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

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 C11 , Bodipy 581/591 , N/A , Invitrogen , D3861.

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 C11 , Bodipy 581/591 , N/A , Invitrogen , D3861.

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 C11 , Bodipy 581/591 , N/A , Invitrogen , D3861.

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 C11 , Bodipy 581/591 , N/A , Invitrogen , D3861.

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 C11 , Bodipy 581/591 , N/A , Invitrogen , D3861.

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 C11 , Bodipy 581/591 , N/A , Invitrogen , D3861.

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

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: Epidermal cells were then incubated with Mitotracker, Mitosox, ER-tracker, Bodipy, or Bodipy-C16 according to the manufacturer’s instructions (Invitrogen) before fixation using 2% PFA in PBS for 15 min at RT.

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: Antibodies and reagents for flow cytometry and immunofluorescence microscopy

Article Snippet: Epidermal cells were then incubated with Mitotracker, Mitosox, ER-tracker, Bodipy, or Bodipy-C16 according to the manufacturer’s instructions (Invitrogen) before fixation using 2% PFA in PBS for 15 min at RT.

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

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 , Bodipy 493/503 , N/A , Invitrogen , D3922.

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 , Bodipy 493/503 , N/A , Invitrogen , D3922.

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 , Bodipy 493/503 , N/A , Invitrogen , D3922.

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 , Bodipy 493/503 , N/A , Invitrogen , D3922.

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 , Bodipy 493/503 , N/A , Invitrogen , D3922.

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 , Bodipy 493/503 , N/A , Invitrogen , D3922.

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