Journal: bioRxiv

Article Title: A long-chain fatty acid elongase Elovl6 regulates mechanical damage–induced keratinocyte death and skin inflammation

doi: 10.1101/264838

Figure Lengend Snippet: (A to D) Representative gross findings (A), histology (hematoxylin and eosin staining) (B), epidermal thickness (C), and numbers of total infiltrating cells and neutrophils in the dorsal skin (D) of wild-type (n =6 or 8) and Elovl6 -/- (n =6 or 8) mice before and on day 9 after the start of tape stripping. (E, F) Epidermal thickness (E) and numbers of total infiltrating cells and neutrophils in the skin (F) of Elovl6 fl/fl (n = 5) and Elovl6 fl/fl K14 -Cre (n = 4) on day 9 after the start of tape stripping. Black bars indicate scale (50 μm) (B). Error bars indicate 1 SD; *, P < 0.05; **, P < 0.01; ***, P < 0.001. Data are representative of three (A-D) and two (E, F) independent experiments.

Article Snippet: Primary mouse keratinocytes were stimulated with bovine HMGB-1 (Chondrex, Redmond, Washington, USA) or mouse IL-1α (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Staining, Stripping Membranes

Journal: bioRxiv

Article Title: A long-chain fatty acid elongase Elovl6 regulates mechanical damage–induced keratinocyte death and skin inflammation

doi: 10.1101/264838

Figure Lengend Snippet: ( A ) The fetuses at E13.5 and E17.5 from WT and Elovl6 -/- mice were stained with 0.1% toluidine blue for 24 h and photographed. ( B ) The transepidermal water loss of 6-8 weeks old WT and Elovl6 -/- mice was measured before and after tape stripping (n = 14). Error bars indicate SD. NS, not significant. Data are representative of three independent experiments.

Article Snippet: Primary mouse keratinocytes were stimulated with bovine HMGB-1 (Chondrex, Redmond, Washington, USA) or mouse IL-1α (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Staining, Stripping Membranes

Journal: bioRxiv

Article Title: A long-chain fatty acid elongase Elovl6 regulates mechanical damage–induced keratinocyte death and skin inflammation

doi: 10.1101/264838

Figure Lengend Snippet: (A) Quantitative RT-PCR analysis of epidermis of wild-type and Elovl6 -/- mice isolated 6 h after tape stripping (n = 10 in each group). (B) Epidermis was isolated before, and 12 h after, tape stripping from wild-type and Elovl6 -/- mice and cultured for 24 h. The concentrations of IL-1β and CXCL-1 in the supernatants were measured by using cytometric bead array (n = 11 per group). (C) Quantitative RT-PCR analysis of ¡lift and Cxcl1 in the epidermis of wild-type, Myd88 -/- , and Ticami -/- mice before, and 6 h after, tape stripping (n = 7 to 11 per group). Error bars indicate 1 SD; *, P < 0.05; ***, P < 0.001. Data are representative of three independent experiments.

Article Snippet: Primary mouse keratinocytes were stimulated with bovine HMGB-1 (Chondrex, Redmond, Washington, USA) or mouse IL-1α (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Quantitative RT-PCR, Isolation, Stripping Membranes, Cell Culture

Journal: bioRxiv

Article Title: A long-chain fatty acid elongase Elovl6 regulates mechanical damage–induced keratinocyte death and skin inflammation

doi: 10.1101/264838

Figure Lengend Snippet: (A) Representative histopathology of wild-type and Elovl6 -/- mice 4 h after tape stripping. Black arrows indicate degenerated keartinocytes with irregularly shaped nuclei. Scale bar, 50 pm. (B, C) Flow cytometry of epidermal cells isolated from the skin of wild-type and Elovl6 -/- mice at the indicated time points after tape stripping. Cells were stained with anti-CD45.2, anti-CD49f and propidium iodide (PI) and the proportion of PI+ cells in CD45.2-CD49f+ cells were shown (n = 6 to 10 per group). Error bars indicate 1 SD; *, P < 0.05. Data are representative of two independent experiments.

Article Snippet: Primary mouse keratinocytes were stimulated with bovine HMGB-1 (Chondrex, Redmond, Washington, USA) or mouse IL-1α (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Histopathology, Stripping Membranes, Flow Cytometry, Isolation, Staining

Journal: bioRxiv

Article Title: A long-chain fatty acid elongase Elovl6 regulates mechanical damage–induced keratinocyte death and skin inflammation

doi: 10.1101/264838

Figure Lengend Snippet: (A) Live cell number of primary peritoneal macrophages 16 h after stimulation with oleic acid (OA) or CVA (300 µM) (n = 3 in each group). (B, E) Primary mouse keratinocytes were cultured for 6 h in the presence or absence of 10 μM of triacsin C (B), or 1 mM necrostatin (Nec-1), 1 mM necrosulfonamide (NSA), 2 mM IM-54, or 1 mM cyclosporine (CyA) (E), followed by stimulation by adding 300 μM CVA; live cells were counted 16 h afterward (n = 3). (C) A representative dead primary keratinocyte induced by stimulation with 300 μM CVA for 10 h under a transmission electron microscope. (D) Immunofluorescence microscopic study of primary keratinocyte 10 h after stimulation with CVA or 6 h after ultraviolet irradiation. Cells were stained with anti-cleaved caspase 9, followed by Alexa Fluor 594-conjugated secondary antibody and DAPI. White bars indicate a scale (20 μm). Percentage of cleaved caspase 9-positive cells was calculated (n = 3). Error bars indicate SD. NS, not significant. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Data are representative of more than two independent experiments.

Article Snippet: Primary mouse keratinocytes were stimulated with bovine HMGB-1 (Chondrex, Redmond, Washington, USA) or mouse IL-1α (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Cell Culture, Transmission Assay, Microscopy, Immunofluorescence, Irradiation, Staining

Journal: bioRxiv

Article Title: A long-chain fatty acid elongase Elovl6 regulates mechanical damage–induced keratinocyte death and skin inflammation

doi: 10.1101/264838

Figure Lengend Snippet: (A) Enzyme-linked immunosorbent assay of HMGB-1 (n = 4 per group) and cytokine bead array of IL-1α (n = 3 per group) in the supernatant of cultured primary keratinocytes 10 h after initiation of stimulation with 300 pM OA or CVA. (B) Quantitative RT-PCR analysis of Il1β and Cxcl1 in the epidermis of wild-type mice 6 h after topical application of ethanol (control) (n = 10) or 15mM of OA (n = 13) or CVA (n = 14) (B). (C, D) Wild-type and Elovl6 -/- mice were treated with PBS (n = 10 and 12, respectively), an IL-1 receptor antagonist (n = 9 and 8, respectively), or a CXCR-2 antagonist (n = 5 and 4, respectively) daily for 9 days, from the beginning on the day of tape stripping. Epidermal thickness (C) and the number of infiltrating neutrophils (D) were analyzed on day 9. (E) A proposed signal pathway from mechanical damage onto the skin to skin inflammation. Error bars indicate SD; *, P < 0.05; **, P < 0.01, ***, P < 0.001; NS, not significant. Data are representative of at least two independent experiments.

Article Snippet: Primary mouse keratinocytes were stimulated with bovine HMGB-1 (Chondrex, Redmond, Washington, USA) or mouse IL-1α (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Quantitative RT-PCR, Stripping Membranes

Journal: bioRxiv

Article Title: A long-chain fatty acid elongase Elovl6 regulates mechanical damage–induced keratinocyte death and skin inflammation

doi: 10.1101/264838

Figure Lengend Snippet: Quantitative RT-PCR analysis of Il1β and Cxcl1 in keratinocytes of wild-type and Elovl6 -/- mice after stimulation or not with HMGB-1 or IL-1α in vitro (n=10 per group) (A) and in the epidermis isolated 4 h after injection intradermally with PBS, HMGB-1, or IL-1α (n = 8 per each group) (B).

Article Snippet: Primary mouse keratinocytes were stimulated with bovine HMGB-1 (Chondrex, Redmond, Washington, USA) or mouse IL-1α (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Quantitative RT-PCR, In Vitro, Isolation, Injection