Journal: The Journal of Clinical Investigation

Article Title: Biallelic GLTP mutations cause nonsyndromic epidermal differentiation disorder via disrupted epidermal glucosylceramide transport

doi: 10.1172/JCI198835

Figure Lengend Snippet: ( A ) Nile Red staining shows dot-like aggregates in the corneocytes of patient 3 (P3) compared with the continuous linear lipid structures observed in the healthy controls (HC). Scale bars: 20 μm. ( B ) Immunofluorescence of glucosylceramide (GlcCer) in the frozen skin section reveals increased GlcCer accumulation within the SG of the patient, in contrast to the presence in SC layer of the HC section. Scale bars: 50 μm. ( C ) Double immunofluorescent staining of GLTP and ceramide in primary human keratinocytes. Under undifferentiated condition, both GLTP and GlcCer/ceramide show cytoplasmic localization. GlcCer/ceramide immunocytochemistry demonstrated congested patterns of ceramide in undifferentiated si- GLTP keratinocytes. Change in localization of GLTP from cytoplasmic appearance to a both membranous and cytoplasmic appearance in response to Ca 2+ -induced differentiation. In contrast with downregulation and cell membranous distribution of ceramide in the si-NC differentiated keratinocytes, GlcCer/ceramide demonstrated accumulation and congested patterns of ceramide in differentiated si- GLTP keratinocytes. Scale bars: 10 μm.

Article Snippet: For GlcCer synthase inhibition, different concentrations of Eliglustat (HY-14885A, MCE) were maintained throughout 3-day differentiation period of keratinocytes induced by 1.3 mM CaCl 2 .

Techniques: Staining, Immunofluorescence, Immunocytochemistry

Journal: The Journal of Clinical Investigation

Article Title: Biallelic GLTP mutations cause nonsyndromic epidermal differentiation disorder via disrupted epidermal glucosylceramide transport

doi: 10.1172/JCI198835

Figure Lengend Snippet: ( A ) Immunofluorescence staining of LC3B and p62 in the epidermis of patients (P1, P2) and HC. Scale bars: 50 μm. ( B ) Western blot of LC3B and p62 protein expression in the epidermis from Gltp +/+ and Gltp –/– mouse newborns. ( C ) Expression levels of autophagic markers LC3B and p62 in si- GLTP and si-NC keratinocytes treated with E64d & Pepstatin A (E&P). ( D ) Representative images of keratinocytes infected with adenovirus Ad-mCherry-GFP-LC3B. Scale bars: 10 μm. ( E ) Quantification analysis of autophagosomes (AP, yellow puncta in merged images) and autolysosomes (AL, red-only puncta in merged images) in keratinocytes. Bars represent the mean ± SEM from 3 independent experiments (40–50 cells in total per group). ( F ) Distribution of C6-NBD-GlcCer (green), a fluorescent GlcCer analog, in differentiated keratinocytes with lysotracker (red) and Hoechst nuclear stain (blue). Scale bars: 10 μm. ( G ) Expression of autophagic markers LC3B and p62 in si- GLTP keratinocytes treated with varying doses of Eliglustat. ( H ) RT-qPCR profiling of differentiation markers IVL , FLG , and LOR in Ca 2+ -induced differentiation keratinocytes treated with Eliglustat. n = 3, ± SEM; **** P < 0.0001 (1-way ANOVA with Dunnett’s multiple comparison test).

Article Snippet: For GlcCer synthase inhibition, different concentrations of Eliglustat (HY-14885A, MCE) were maintained throughout 3-day differentiation period of keratinocytes induced by 1.3 mM CaCl 2 .

Techniques: Immunofluorescence, Staining, Western Blot, Expressing, Infection, Quantitative RT-PCR, Comparison

Journal: The Journal of Biological Chemistry

Article Title: Ceramide-CD300f Binding Inhibits Lipopolysaccharide-induced Skin Inflammation *

doi: 10.1074/jbc.M116.768366

Figure Lengend Snippet: Ceramide-CD300f binding inhibited the release of chemical mediators from LPS-stimulated mast cells and neutrophils in vitro . A and B , the levels of MIP2 or LTC4 released from WT or CD300f −/− BMMC ( A ) or neutrophils ( B ) stimulated with 100 ng/ml LPS for 6 h. Cells were preincubated on plates coated with ceramide, PC, or vehicle for 1 h before stimulation. Data are representative of three independent experiments. Means ± S.D. are plotted. *, p < 0.01 (Student's t test).

Article Snippet: Cytokines were obtained from R&D Systems, and C-24 ceramide was obtained from Toronto Research Chemicals, Inc. 1,2-Dipalmitoyl- sn -glycero-3-phosphocholine (PC) was obtained from Echelon Biosciences Inc.

Techniques: Binding Assay, In Vitro

Journal: The Journal of Biological Chemistry

Article Title: Ceramide-CD300f Binding Inhibits Lipopolysaccharide-induced Skin Inflammation *

doi: 10.1074/jbc.M116.768366

Figure Lengend Snippet: Ceramide-CD300f binding inhibits LPS-induced skin inflammation. A and B , dye extravasation in ears of WT and CD300f −/− mice 1 h after intradermal injection with LPS. OD , optical density. C and D , the number of neutrophils recruited to skin pouches of WT and CD300f −/− mice 4 h after intradermal injection with LPS. A–D , ,ice intradermally injected with CD300f-Fc or control Fc ( A ), ceramide antibody or control antibody ( B and C ), or vesicles containing ceramide, PC, or vehicle ( D ) 1 h before LPS injection. Data are representative of two independent experiments. Means ± S.D. are plotted. *, p < 0.01 (Student's t test).

Article Snippet: Cytokines were obtained from R&D Systems, and C-24 ceramide was obtained from Toronto Research Chemicals, Inc. 1,2-Dipalmitoyl- sn -glycero-3-phosphocholine (PC) was obtained from Echelon Biosciences Inc.

Techniques: Binding Assay, Injection, Control