Journal: iScience

Article Title: OBP2A regulates epidermal barrier function and protects against cytotoxic small hydrophobic molecules

doi: 10.1016/j.isci.2024.111093

Figure Lengend Snippet:

Article Snippet: Membrane staining was performed by using antibodies to OBP2A (orb582625, Biorbyt Ltd., Cambridge, UK), FABP5 (39926, Cell Signaling Technology, MA, USA), PERK (5683, Cell Signaling Technology, MA, USA), Bip (3177, Cell Signaling Technology, MA, USA), PDI (3501, Cell Signaling Technology, MA, USA), involucrin (ab53112, abcam, Cambridge, UK), keratin 10 (ab76318, abcam, Cambridge, UK), corneodesmosin (13184, proteintech, IL, USA), desmoglein 2 (MAB947, R&D Systems, MO, USA), and β-actin (sc-47778, Santa Cruz Biotechnology, TX, USA) as primary antibodies and a Novex AP Rabbit Chemiluminescent Detection Kit or Novex AP Mouse Chemiluminescent Detection Kit (Invitrogen, CA, USA) as secondary antibodies.

Techniques: Imaging, Recombinant, Transfection, Saline, Sterility, Cell Culture, Protease Inhibitor, DC Protein Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Quantitation Assay, Fluorescence, Sequencing, Software

Journal: iScience

Article Title: OBP2A regulates epidermal barrier function and protects against cytotoxic small hydrophobic molecules

doi: 10.1016/j.isci.2024.111093

Figure Lengend Snippet:

Article Snippet: For immunostaining of OBP2A, filaggrin, involucrin, keratin 10, transglutaminase 1, claudin 1, corneodesmosin, and desmoglein 2, antibodies to OBP2A (orb37374, Biorbyt Ltd., Cambridge, UK), filaggrin (sc-66192, Santa Cruz Biotechnology, TX, USA), involucrin (ab53112, abcam, Cambridge, UK), keratin 10 (20R-CP002, Fitzgerald Industries International, MA, USA), transglutaminase 1 (sc-166467, Santa Cruz Biotechnology, TX, USA), claudin 1 (71–7800, Invitrogen, CA, USA), corneodesmosin (ab204235, abcam, Cambridge, UK), and desmoglein 2 (MAB947, R&D Systems, MO, USA) were used as primary antibodies, and donkey anti-rabbit IgG (Invitrogen, CA, USA) or donkey anti-guinea pig IgG (Jackson ImmunoResearch, PA, USA) was used as a second antibody.

Techniques: Imaging, Recombinant, Transfection, Saline, Sterility, Cell Culture, Protease Inhibitor, DC Protein Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Quantitation Assay, Fluorescence, Sequencing, Software

Journal: Cell Death & Disease

Article Title: Desmoglein-2 is important for islet function and β-cell survival

doi: 10.1038/s41419-022-05326-2

Figure Lengend Snippet: A Immunofluorescence confocal microscopy of human pancreas from a healthy body donor stained for insulin (green), DSG2 (red), and nuclei (blue). Scale bar = 20 μm. Insert top right is representative of isotype control stains. B Surface expression of DSG2 by flow cytometric analysis on freshly isolated human islet cells from healthy donors labelled with Newport Green (NPG) dye identifying β-cells, isotype control (dotted line), and DSG2 (solid line); with all single cells gated from a live population (7-AAD). C Immunofluorescence microscopy of partly digested human islets from a healthy donor stained for β-cells by labelling for insulin (green), DSG2 (red), and nuclei (blue). Scale bar = 10 μm. Insert top right is representative of isotype control stains. D Microarray gene expression of insulin ( INS , green), desmogleins (DSG1-4 , red), and desmocollins (DSC1-3 , purple) in isolated islet preparations from 9 healthy human body donors. Data represented as the average log2 expression ± SEM with a threshold cut off of 5. E Complete RNA sequencing data from 188 human islets expressed as log2 FPKM (Fragments Per Kilobase Million, value of 1 noted in blue line) with ranked expression of DSG2 (red line) compared to insulin ( INS , grey line) and potassium channel ( KCNJ1 , grey line).

Article Snippet: Sections were probed with the following antibodies: guinea pig anti-human/mouse insulin pAb, mouse anti-mouse glucagon mAb, and rat anti-mouse somatostatin mAb (all Abcam), anti-DSG2 mAbs (clone AF947 R&D Systems, and clone 10D2 (gift, James K Wahl III)), rabbit anti-E-cadherin pAb (Cell Signalling Technologies, Danvers, MA, USA) and isotype controls (e.g. IgG (Abcam or R&D Systems) or anti-maltose clone 12B12, gift, James K Wahl III).

Techniques: Immunofluorescence, Confocal Microscopy, Staining, Control, Expressing, Isolation, Microscopy, Microarray, RNA Sequencing Assay

Journal: Cell Death & Disease

Article Title: Desmoglein-2 is important for islet function and β-cell survival

doi: 10.1038/s41419-022-05326-2

Figure Lengend Snippet: Immunofluorescence confocal microscopy of A human and B mouse islets stained for insulin (green), DSG2 (red), somatostatin (magenta), and nuclei (DAPI, blue). Insets with arrow labelled ‘1’ indicating insulin-positive cells, arrow labelled ‘2’ indicating somatostatin positive cells, and cells with neither insulin nor somatostatin staining indicated with arrow labelled ‘3’. Scale bar = 50 μm. Insert top right is representative of isotype control stains. C Mouse pancreata were isolated from wildtype (WT, black circles) and Dsg2 lo/lo (blue squares) mice and Dsg2 gene expression determined via qRT-PCR. Data are expressed as mean ± SEM relative to housekeeper gene ( Hprt1 ), n = 4 mice per group, * p < 0.05 vs WT. D Immunofluorescence confocal microscopy of pancreas sections from WT and Dsg2 lo/lo mice stained for insulin (green), DSG2 (red), and nuclei (blue). Red arrow indicating blood vessels. Scale bar = 50 μm.

Article Snippet: Sections were probed with the following antibodies: guinea pig anti-human/mouse insulin pAb, mouse anti-mouse glucagon mAb, and rat anti-mouse somatostatin mAb (all Abcam), anti-DSG2 mAbs (clone AF947 R&D Systems, and clone 10D2 (gift, James K Wahl III)), rabbit anti-E-cadherin pAb (Cell Signalling Technologies, Danvers, MA, USA) and isotype controls (e.g. IgG (Abcam or R&D Systems) or anti-maltose clone 12B12, gift, James K Wahl III).

Techniques: Immunofluorescence, Confocal Microscopy, Staining, Control, Isolation, Expressing, Quantitative RT-PCR

Journal: Cell Death & Disease

Article Title: Desmoglein-2 is important for islet function and β-cell survival

doi: 10.1038/s41419-022-05326-2

Figure Lengend Snippet: A Immunohistochemistry of pancreata harvested from WT and Dsg2 lo/lo mice stained with haematoxylin and eosin to identify islet clusters (black dotted outline) within the exocrine tissue. Scale bar = 100 µm. B Numbers of islets quantified from three entire sections across the organ for WT ( n = 3 mice) and Dsg2 lo/lo ( n = 4 mice). Data are expressed as mean ± SEM, ** p < 0.01 vs WT. C Islet area determined using ImageJ and presented in arbitrary units for the 60 islets assessed from 3 WT mice and 47 islets assessed from 4 Dsg2 lo/lo mice. Data are expressed as mean ± SEM, * p < 0.05 vs WT. D Representative images of immunofluorescence staining on pancreas sections from WT and Dsg2 lo/lo mice to identify insulin producing β-cells (red), glucagon-producing α-cells (blue), and somatostatin-producing δ-cells (green). Insert top left is representative of isotype control stains. Scale bar = 50 μm, fluorescence staining quantified as pixel intensity for insulin ( E ), glucagon ( F ), and somatostatin ( G ). Data are expressed as mean pixel intensity ± SEM, for the 38 islets assessed from 3 WT mice and 33 islets assessed from 4 Dsg2 lo/lo mice, * p < 0.05 vs WT. H Representative images of immunohistochemistry staining on pancreas sections from WT and Dsg2 lo/lo mice to identify blood vessels (CD31+). Scale bar = 100 μm. Insert top left is representative of isotype control stains. I % CD31 + vessels per islet quantified for the 35 islets assessed from 4 WT mice and 19 islets assessed from 4 Dsg2 lo/lo mice, data are expressed as mean ± SEM.

Article Snippet: Sections were probed with the following antibodies: guinea pig anti-human/mouse insulin pAb, mouse anti-mouse glucagon mAb, and rat anti-mouse somatostatin mAb (all Abcam), anti-DSG2 mAbs (clone AF947 R&D Systems, and clone 10D2 (gift, James K Wahl III)), rabbit anti-E-cadherin pAb (Cell Signalling Technologies, Danvers, MA, USA) and isotype controls (e.g. IgG (Abcam or R&D Systems) or anti-maltose clone 12B12, gift, James K Wahl III).

Techniques: Immunohistochemistry, Staining, Immunofluorescence, Control, Fluorescence

Journal: Cell Death & Disease

Article Title: Desmoglein-2 is important for islet function and β-cell survival

doi: 10.1038/s41419-022-05326-2

Figure Lengend Snippet: A Immunofluorescence confocal microscopy of a WT mouse pancreatic islet stained for DSG2 with DSG2+ pancreatic islet (demarked in green) and DSG2+ blood vessel (demarked in red). B Transmission electron microscopy (TEM) of pancreatic islets in WT and Dsg2 lo/lo mice showing the vasculature endothelial cells (EC) with the lumen on one side and the β-cell on the other. Arrows indicate the EC fenestrations and the insert below shows the sieve plates of fenestrae counted per μm length of vessel lining from n = 6–16 islet-associated vessels from 2 mice per group. **** p < 0.0001 vs WT, scale bar = 1 μm. C Anaesthetised mice (WT and Dsg2 lo/lo ) were injected i.v. with 70 kDa FITC-Dextran prior to intravital 2-photon microscopy of the ear. Snapshots of 0 and 15 min time points were quantified via mean normalised fluorescence of Dextran signal ± SEM ( n = 5–6 mice), * p < 0.05 vs WT at 15 min. D arrows identifying the membranes encasing the insulin-containing granules with their typical electron-dense core. Scale bar = 1 μm.

Article Snippet: Sections were probed with the following antibodies: guinea pig anti-human/mouse insulin pAb, mouse anti-mouse glucagon mAb, and rat anti-mouse somatostatin mAb (all Abcam), anti-DSG2 mAbs (clone AF947 R&D Systems, and clone 10D2 (gift, James K Wahl III)), rabbit anti-E-cadherin pAb (Cell Signalling Technologies, Danvers, MA, USA) and isotype controls (e.g. IgG (Abcam or R&D Systems) or anti-maltose clone 12B12, gift, James K Wahl III).

Techniques: Immunofluorescence, Confocal Microscopy, Staining, Transmission Assay, Electron Microscopy, Injection, Microscopy, Fluorescence

Journal: Cell Death & Disease

Article Title: Desmoglein-2 is important for islet function and β-cell survival

doi: 10.1038/s41419-022-05326-2

Figure Lengend Snippet: A Baseline blood glucose levels (BGL) in WT and Dsg2 lo/lo mice. Results are mean ± SEM, n = 3–4 mice per group. B Glucose tolerance in WT and Dsg2 lo/lo mice following i.v. injection of 1 g/kg glucose with BGLs measured at 0 (dotted line), 2.5, 5, 15, and 30 min post injection. Data are expressed as mean ± SEM from n = 7–9 individual mice per group. C Islets isolated from WT or Dsg2 lo/lo mice tested for glucose-stimulated insulin release at low glucose (2 mM) and then high glucose (20 mM) for 1 h, represented as mean ± SEM of insulin release to DNA, n = 4–7 mice per group. D Cytokine induced apoptosis of islets isolated from WT and Dsg2 lo/lo mice. Islets were exposed to TNFα, IL-1β and IFNγ for 72 h prior to staining for with Annexin V and propidium iodide (PI) to assess cell death, n = 4–6 mice per group and 3 separate experiments, * p < 0.05 vs WT.

Article Snippet: Sections were probed with the following antibodies: guinea pig anti-human/mouse insulin pAb, mouse anti-mouse glucagon mAb, and rat anti-mouse somatostatin mAb (all Abcam), anti-DSG2 mAbs (clone AF947 R&D Systems, and clone 10D2 (gift, James K Wahl III)), rabbit anti-E-cadherin pAb (Cell Signalling Technologies, Danvers, MA, USA) and isotype controls (e.g. IgG (Abcam or R&D Systems) or anti-maltose clone 12B12, gift, James K Wahl III).

Techniques: Injection, Isolation, Staining

Journal: Cell Death & Disease

Article Title: Desmoglein-2 is important for islet function and β-cell survival

doi: 10.1038/s41419-022-05326-2

Figure Lengend Snippet: A WT and Dsg2 lo/lo mice administered STZ (185 mg/kg) were monitored daily for BGLs. A BGL ≥ 16 mmol/L (black dotted line) indicates the diabetic cut off value with the grey shaded box indicating a normal BGL range. Results are mean ± SEM, n = 8–9 mice per group, * p < 0.05 & ** p < 0.01 vs WT. Area under the curve quantified and presented as mean ± SEM, n = 8–9 mice per group, ** p < 0.01 vs WT. B From A, percentage of mice that became diabetic over time, *p < 0.05 vs WT. C Diabetic C57Bl6/N control (WT, n = 9) mice were transplanted with marginal islet mass of 200 islets harvested from WT ( n = 5) or Dsg2 lo/lo ( n = 4) mice under the kidney capsule. BGLs in individual mice were recorded daily and up to 35 days post-transplantation. ** p < 0.01 vs day 0 BGL, **** p < 0.0001 vs day 0 BGL. D From C , percentage cure of diabetic mice transplanted with marginal mass of islets displayed as Kaplan–Meier curve, where two consecutive readings of ≤11.1 mmol/L was considered a cured mouse.

Article Snippet: Sections were probed with the following antibodies: guinea pig anti-human/mouse insulin pAb, mouse anti-mouse glucagon mAb, and rat anti-mouse somatostatin mAb (all Abcam), anti-DSG2 mAbs (clone AF947 R&D Systems, and clone 10D2 (gift, James K Wahl III)), rabbit anti-E-cadherin pAb (Cell Signalling Technologies, Danvers, MA, USA) and isotype controls (e.g. IgG (Abcam or R&D Systems) or anti-maltose clone 12B12, gift, James K Wahl III).

Techniques: Control, Transplantation Assay

Journal: Cell Death & Disease

Article Title: Desmoglein-2 is important for islet function and β-cell survival

doi: 10.1038/s41419-022-05326-2

Figure Lengend Snippet: A Representative qRT-PCR showing Dsg2 gene expression for siCtrl (black) and siDSG2 (A-C, blue) groups normalised to housekeeper Hprt1 , n = 7 independent experiments, *** p < 0.001. B Cell cycle distribution (G0/G1, S or G2 phase) of Beta-TC-6 cells without (siCtrl, black) and with Dsg2 knockdown (siDSG2, blue) using flow cytometry PI staining, n = 3 independent experiments. C Representative immunofluorescence image of Phalloidin-labelled filamentous actin in Beta-TC-6 cells without (siCtrl, black) and with Dsg2 knockdown (siDSG2-A, blue). Yellow rectangle highlights the area of interest which was used to calculate the mean grey value (pixels) across the cell from border to border. The mean grey value for siCtrl (black) and siDSG2-A (blue) was converted to area under the curve (AUC), n = 4 independent experiments, ** p < 0.01. D Cytokine/chemokine array of supernatants harvested from Beta-TC-6 cells without (siCtrl) or with Dsg2 knockdown (siDSG2-A), n = 1 experiment. Mean grey value of duplicate dots was calculated and summarised as a bar graph below. White box = positive control, black box = negative control, green box = CXCL10, blue box = TNF-alpha, red box = CXCL12. E Cytokine/chemokine array of supernatants harvested from Beta-TC-6 cells without (siCtrl) or with Dsg2 knockdown (siDSG2-A) following TNFα treatment (100 ng/ml, 24 h), n = 1 experiment. For detectable proteins, mean grey value of duplicate dots was calculated and graphed. White box = positive control, black box = negative control, green box = CXCL10, yellow box = CXCL1, blue box = TNFα, orange box = CCL2, purple box = CXCL2, red box = CXCL12.

Article Snippet: Sections were probed with the following antibodies: guinea pig anti-human/mouse insulin pAb, mouse anti-mouse glucagon mAb, and rat anti-mouse somatostatin mAb (all Abcam), anti-DSG2 mAbs (clone AF947 R&D Systems, and clone 10D2 (gift, James K Wahl III)), rabbit anti-E-cadherin pAb (Cell Signalling Technologies, Danvers, MA, USA) and isotype controls (e.g. IgG (Abcam or R&D Systems) or anti-maltose clone 12B12, gift, James K Wahl III).

Techniques: Quantitative RT-PCR, Expressing, Knockdown, Flow Cytometry, Staining, Immunofluorescence, Positive Control, Negative Control

Journal: PLoS ONE

Article Title: Effective Apical Infection of Differentiated Human Bronchial Epithelial Cells and Induction of Proinflammatory Chemokines by the Highly Pneumotropic Human Adenovirus Type 14p1

doi: 10.1371/journal.pone.0131201

Figure Lengend Snippet: Confocal microscopy immunofluorescence analysis of differentiated human bronchial epithelial cells. Figs A, B, C show XY planes, Figs D, E, F show XZ planes. (A, D) Cells were infected from the apical side with HAdV-B14p1 at a moi of 10 (TCID 50 /cell), fixated 4 days p.i. and stained for HAdV in green (FITC conjugated antibody) and the desmoglein 2 (DSG2 receptor) in red (dsRed antibody), the nucleus was counterstained in blue (DAPI). (B, E) Differentiated human bronchial epithelial cells stained for DSG2 receptor in red (dsRed) and occludin (OCLN) a tight junction marker in green (FITC), nucleus was counterstained in blue (DAPI). (C, F) Differentiated human bronchial epithelial cells were stained for CAR receptor in red (dsRed) and the tight junction marker OCLN in green (FITC) and the nucleus in blue (DAPI).

Article Snippet: Primary antibodies used were a polyclonal rabbit occludin (OCLN) antibody (1:40 diluted; Invitrogen, Paisley, UK), a monoclonal mouse CAR antibody (RmcB, 1:100 diluted, kindly provided by M. Bergelson), a monoclonal mouse desmoglein 2 (DSG2) antibody (Clone 6D8, 1:50 diluted, Santa Cruz Biotechnology, Santa Cruz, CA), and a FITC conjugated monoclonal adenovirus antibody (Millipore, Bilerica, MA).

Techniques: Confocal Microscopy, Immunofluorescence, Infection, Staining, Marker

Journal: bioRxiv

Article Title: Catalytic antibodies in arrhythmogenic cardiomyopathy patients cleave desmoglein 2 and N-cadherin and impair cardiomyocyte cohesion

doi: 10.1101/2023.02.08.527624

Figure Lengend Snippet: Detection of antibodies against ICD proteins in human left ventricular tissue by immunofluorescence analysis. Human left ventricular tissue was incubated with respective AC-IgGs and Control-IgGs as indicated in the figure. N-CAD was used as a marker for ICDs, and presence of anti-ICD proteins was defined when there was an overlap of IgG staining with N-CAD (white arrows). The yellow arrow in control-IgG shows no positive staining of ICDs. Anti-DSG2 antibody was used to show the presence of DSG2 in the cardiac tissue used. AC patients were grouped into DPC (harboring desmoplakin gene mutations) and ARVC (harboring plakophilin2 gene mutations). Images represent immunofluorescence analysis of 3 repeats. Scale bar: 10 µm.

Article Snippet: The next day, after being washed 4 times with PBS, the wells were incubated with either goat anti-mouse IgG-HRP (Dianova, #115-035-068) at 1:2000 dilution in blocking buffer for DSG2-Origene antibody and DSG2-Abcam antibody, or goat anti-human IgG-HRP (AffiniPure F(ab’)□ Fragment Goat Anti-Human IgG (H+L) Pox; Jackson Immuno Research # 109-036-088) at 1:100000 dilution in blocking buffer for the patient samples.

Techniques: Immunofluorescence, Incubation, Marker, Staining

Journal: bioRxiv

Article Title: Catalytic antibodies in arrhythmogenic cardiomyopathy patients cleave desmoglein 2 and N-cadherin and impair cardiomyocyte cohesion

doi: 10.1101/2023.02.08.527624

Figure Lengend Snippet: In vitro DSG2 and N-CAD Cleavage assays A. Human DSG2-Fc protein (200ng/lane) or B. N-CAD-Fc protein (200 ng/lane) was incubated with respective IgGs from AC patients (AC1-AC15), water (H 2 O), healthy control IgG (IgG), PV-IgG and AK23 (murine pemphigus monoclonal anti-DSG3 antibody, as a negative control) for 4 hours, without (C) and with protease inhibitor (I, cOmplete™) and Western blot analysis was performed. A representative image of 3 experimental repeats is shown. # Indicates the cleaved fragments. * Indicates mouse IgG heavy chain.

Article Snippet: The next day, after being washed 4 times with PBS, the wells were incubated with either goat anti-mouse IgG-HRP (Dianova, #115-035-068) at 1:2000 dilution in blocking buffer for DSG2-Origene antibody and DSG2-Abcam antibody, or goat anti-human IgG-HRP (AffiniPure F(ab’)□ Fragment Goat Anti-Human IgG (H+L) Pox; Jackson Immuno Research # 109-036-088) at 1:100000 dilution in blocking buffer for the patient samples.

Techniques: In Vitro, Incubation, Negative Control, Protease Inhibitor, Western Blot

Journal: bioRxiv

Article Title: Catalytic antibodies in arrhythmogenic cardiomyopathy patients cleave desmoglein 2 and N-cadherin and impair cardiomyocyte cohesion

doi: 10.1101/2023.02.08.527624

Figure Lengend Snippet: Homophilic DSG2 and N-CAD interaction probabilities measured by AFM A . Schematic presentation of an AFM interaction experiment in cell-free conditions. Recombinant DSG2/N-CAD extracellular domain containing proteins tagged with Fc fragments were covalently linked via a PEG linker to the AFM tip and the mica sheet. A laser is directed to the cantilever tip and reflected onto a photodetector. Each interaction event leads to a deflection of the cantilever, detected by the laser reflected on the photodetector and a force-distance curve of a specific binding event was produced, together with a topography image and adhesion events. Quantification of DSG2 binding frequency expressed in relative interaction probability. B . in desmoplakin mutated AC patients (DPC), C . plakophilin 2 mutated patients (ARVC). D . Quantification of N-CAD binding frequency expressed in relative interaction probability in DPC. Each data point represents 1000 curves analyzed across two areas (10 µm x 10 µm). Data are presented as mean ± SEM.* p ≤ 0.05 compared to IgG, and NS is not significant compared to IgG. One-way ANOVA with Holm-Šídák’s multiple comparisons test was performed. N=3-5.

Article Snippet: The next day, after being washed 4 times with PBS, the wells were incubated with either goat anti-mouse IgG-HRP (Dianova, #115-035-068) at 1:2000 dilution in blocking buffer for DSG2-Origene antibody and DSG2-Abcam antibody, or goat anti-human IgG-HRP (AffiniPure F(ab’)□ Fragment Goat Anti-Human IgG (H+L) Pox; Jackson Immuno Research # 109-036-088) at 1:100000 dilution in blocking buffer for the patient samples.

Techniques: Recombinant, Binding Assay, Produced