mab against dsg2  (R&D Systems)

Journal: Molecular oncology

Article Title: Desmoglein-2 expression is an independent predictor of poor prognosis patients with multiple myeloma.

doi: 10.1002/1878-0261.13055

Figure Lengend Snippet: Fig. 1. DSG2 is expressed by MM PC at the gene and protein level in a distinct subset of MM patients. (A, B) In silico analysis of publicly available microarray datasets E-MTAB-363 (A) and E-GEOD-16122 (B) was performed. In these studies, RNA was extracted from CD138+

Article Snippet: Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. with primary mAb against DSG2 (0.9 μg mL 1 final concentration, clone #141409, MAB947 R&D Systems, Minneapolis, MN, USA), anti-CD138 (clone MI15; Dako, 1 : 100 dilution from stock), anti-CD31 (clone 89C2; Cell Signaling Technology, Danvers, MA, USA, 1 : 1200 dilution from stock) or an isotype-matched (IgG1) control antibody (0.5 μg mL 1, Abcam, Cambridge, UK), followed by reaction with DAB, counterstaining using Mayer’s haematoxylin and mounting in DPX.

Techniques: In Silico, Microarray

Journal: Molecular oncology

Article Title: Desmoglein-2 expression is an independent predictor of poor prognosis patients with multiple myeloma.

doi: 10.1002/1878-0261.13055

Figure Lengend Snippet: Fig. 2. DSG2 expression in a subset of MM cell lines. (A) DSG2 gene expression values for 65 human MM cell lines were extracted from a publicly available RNAseq dataset as described in Materials and methods. Cell lines were ranked according to level of DSG2 gene expression for simplicity of visualization. (B, C) For nine of the cell lines shown in A, surface expression of DSG2 protein was assessed by flow cytometry. Examples of negative, low and high expression are shown in (B), while the relationship between gene and surface protein for all cell lines analysed is shown in C (Spearman’s correlation coefficient r = 0.65).

Article Snippet: Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. with primary mAb against DSG2 (0.9 μg mL 1 final concentration, clone #141409, MAB947 R&D Systems, Minneapolis, MN, USA), anti-CD138 (clone MI15; Dako, 1 : 100 dilution from stock), anti-CD31 (clone 89C2; Cell Signaling Technology, Danvers, MA, USA, 1 : 1200 dilution from stock) or an isotype-matched (IgG1) control antibody (0.5 μg mL 1, Abcam, Cambridge, UK), followed by reaction with DAB, counterstaining using Mayer’s haematoxylin and mounting in DPX.

Techniques: Expressing, Gene Expression, Cytometry

Journal: Molecular oncology

Article Title: Desmoglein-2 expression is an independent predictor of poor prognosis patients with multiple myeloma.

doi: 10.1002/1878-0261.13055

Figure Lengend Snippet: Fig. 3. DSG2 expression in MM is strongly associated with reduced survival, independent of NSD2. (A) Microarray dataset GSE4581 was analysed for expression of DSG2 using probe set 1553105. Visual inspection of the data spread revealed a cluster of samples with elevated DSG2 expression. A 70/30 percentile split was applied to the data, which cleanly separated these DSG2-low and DSG2-high populations, as shown, for further analysis. (B) Overall survival was compared between the DSG2-low (lower 70%, n = 289) and DSG2-high (upper 30%, n = 125) subsets using Kaplan–Meier analysis. P < 0.01 (C) Expression of DSG2 was compared between patients grouped into disease subtypes according to gene expression signatures. DSG2 expression was significantly greater in the MS subset compared to all others (Kruskal–Wallis test). (D, E) Scatterplots comparing expression of DSG2 and NSD2 genes in all samples (D) or non-MS samples only (E). Dotted lines indicate thresholds for expression based on 70th percentile (DSG2) or 80th percentile (NSD2). Values represent the number of samples in each quadrant. (F) The non-MS patient cohort was stratified into DSG2-low and DSG2-high subsets and overall patient survival compared using Kaplan–Meier analysis.

Article Snippet: Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. with primary mAb against DSG2 (0.9 μg mL 1 final concentration, clone #141409, MAB947 R&D Systems, Minneapolis, MN, USA), anti-CD138 (clone MI15; Dako, 1 : 100 dilution from stock), anti-CD31 (clone 89C2; Cell Signaling Technology, Danvers, MA, USA, 1 : 1200 dilution from stock) or an isotype-matched (IgG1) control antibody (0.5 μg mL 1, Abcam, Cambridge, UK), followed by reaction with DAB, counterstaining using Mayer’s haematoxylin and mounting in DPX.

Techniques: Expressing, Microarray, Gene Expression

Journal: Molecular oncology

Article Title: Desmoglein-2 expression is an independent predictor of poor prognosis patients with multiple myeloma.

doi: 10.1002/1878-0261.13055

Figure Lengend Snippet: Fig. 4. Differential gene expression analysis comparing DSG2-low and DSG2-high subsets. Dataset GSE4581 was stratified into DSG2-low (blue bar) and DSG2-high (red bar) patient subsets as per Fig. 3, and genes differentially expressed between the two groups were identified and displayed in heatmaps. Clustering of genes displayed in the heatmap was unsupervised and shown as analyses of the entire patient cohort (A), or only the subgroup of patients lacking MMSET expression (MS-neg; B).

Article Snippet: Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. with primary mAb against DSG2 (0.9 μg mL 1 final concentration, clone #141409, MAB947 R&D Systems, Minneapolis, MN, USA), anti-CD138 (clone MI15; Dako, 1 : 100 dilution from stock), anti-CD31 (clone 89C2; Cell Signaling Technology, Danvers, MA, USA, 1 : 1200 dilution from stock) or an isotype-matched (IgG1) control antibody (0.5 μg mL 1, Abcam, Cambridge, UK), followed by reaction with DAB, counterstaining using Mayer’s haematoxylin and mounting in DPX.

Techniques: Gene Expression, Expressing

Journal: The Journal of cell biology

Article Title: DPM1 modulates desmosomal adhesion and epidermal differentiation through SERPINB5.

doi: 10.1083/jcb.202305006

Figure Lengend Snippet: Figure 2. DPM1 modulates DSP localization and cytoskeletal organization. (a) Immunostaining of DSP and DSG2 in HaCaT keratinocytes. Merge indicates overlap between DSP, DSG2, and nuclei stained with DAPI. Scale bar: 10 µm distance. (b) Quantification of the number of DSP puncta over the respective length of cell membrane (µm) from individual cells (represented by individual dots), N = 3. Unpaired Student’s t test. (c) Immunostaining of DSP and DSG2 in primary human keratinocytes. Scale bar: 10 µm distance. Panel shows representatives of three biological replicates. (d) Immunostaining of DSP in 3D-RHE of control (sgNT1) and sgDPM1 conditions. White dashed line indicates insert membrane. Magenta dashed rectangles mark regions magnified on the right (zoomed 2× of original image). Representative of three biological replicates. Scale bar: 10 µm distance. (e–g) Keratin staining depicted by pan-cytokeratin in

Article Snippet: The following primary antibodies were diluted with odyssey blocking buffer in trisbuffered-saline containing 0.1% Tween20 (Thermo Fisher Scientific) and incubated overnight at 4°C, with rotation: mouse Dsc2 mAb (#60239-1-Ig; Proteintech), mouse Dsg2 mAb (clone 10G11, #BM5016; Acris), rabbit Dsg3 pAb (EAP3816; Elabscience), mouse Pkp1 mAb (clone 10B2, #sc-33636; Santa Cruz), mouse Pkp2 mAb (#651101; Progen), mouse PG mAb (clone PG5.1, #61005; Progen), mouse DSP mAb (#sc-390975; Santa Cruz), mouse E-Cad mAb (clone 36, #610181; BD Biosciences), rabbit DSC1 mAb (#ab150382; Abcam), mouse DSG1 (#651111; Progen), rabbit keratin 10 (#905403; BioLegend), mouse keratin 14 (ab7800; Abcam), rabbit DPM1 (#12403-2-AP; Proteintech), rabbit SERPINB5 (MASPIN #ab182785; Abcam), rabbit mGFP (#TA150122; Thermo Fisher Scientific), mouse GAPDH mAb (clone 0411, #sc-47724; Santa Cruz), and mouse α-tubulin (clone 10D8, #627901; BioLegend).

Techniques: Immunostaining, Staining, Membrane, Control

Journal: PLoS ONE

Article Title: Desmoglein 2 Is Less Important than Desmoglein 3 for Keratinocyte Cohesion

doi: 10.1371/journal.pone.0053739

Figure Lengend Snippet: (A) Immunofluorescence staining of cryosections of human skin demonstrating expression of Desmoglein (Dsg) 2 and Dsg3 in epidermis. For Dsg2 also the expression in the hair follicle is shown. Scale bar, 20 µm. (B) Immunofluorescence staining of Dsg2 and Dsg3 in a human keratinocyte cell line (HaCaT). Cells were cultured for 4 d in high Ca 2+ -medium. Scale bar, 20 µm. (C) Western blots analysis of Dsg2 and Dsg3 in confluent 4 d HaCaT cells. ß-actin was used as loading control.

Article Snippet: Following primary antibodies were used to detect proteins by immunostaining and/or Western blot analysis: anti-Dsg1 (clone P124, Progen, Heidelberg, Germany), anti-Dsg2 mAb (clone 10G11, Progen, custom-made without any preservation components), anti-Dsg3 pAb (clone H-145, Santa Cruz Biotechnology, Santa Cruz, California), anti-Dsg3 mAb (clone 5G11, Life Technologies, Carlsbad, California), anti-Dsc1 pAb (clone L-15, Santa Cruz Biotechnology), anti-Dsc2 pAb (Progen), anti-Dsc3 mAb (clone U114, Progen), anti-ß-Actin mAb (Sigma, St.Louis, USA), anti-E-Cadherin mAb (clone 36, BD Biosciences), anti-Desmoplakin mAb (Epitomics, California, USA), anti-α-Tubulin mAb (Abcam, Cambridge, UK).

Techniques: Immunofluorescence, Staining, Expressing, Cell Culture, Western Blot

Journal: PLoS ONE

Article Title: Desmoglein 2 Is Less Important than Desmoglein 3 for Keratinocyte Cohesion

doi: 10.1371/journal.pone.0053739

Figure Lengend Snippet: (A) After antibody incubation for 24 h with either a monoclonal Dsg2 antibody (Dsg2 mAb) or AK23, confluent HaCaT monolayers were subjected to dispase-based dissociation assays. Loss of cell cohesion was detectable in cells incubated with AK23 only. (n = 8; * p<0.05 vs. control) Photos were taken immediately after assay performance. 1 hour incubation with 5 mM EGTA led to profound loss of cell cohesion indicating the non-hyperadhesive state of HaCaT cells used for these experiments. (n = 6) (B) Exposing the cells to more severe mechanical stress in dissociation assays increased fragment numbers after AK23 treatment only. (n = 6; * p<0.05 vs. control) (C) Atomic force microscopy was used to demonstrate antibody-mediated interference with homophilic Dsg2 and Dsg3 binding. Both antibodies reduced the binding frequency of their respective antigens. (>1000 force distance cycles on more than 2 different cantilever/substrate combinations; * p<0.05 vs. control) (D) Dispase-based dissociation assay performed with confluent Caco-2 cells after 24 h incubation with Dsg2 mAb showed a significant increase in fragment numbers compared to control cells. (n≥20; * p<0.05 vs. control) (E) Protein expression of Dsg2 in Caco-2 cells was proven by immunofluorescence (scale bar, 20 µm) and (F) Western blot analysis. ß-actin was used as loading control.

Article Snippet: Following primary antibodies were used to detect proteins by immunostaining and/or Western blot analysis: anti-Dsg1 (clone P124, Progen, Heidelberg, Germany), anti-Dsg2 mAb (clone 10G11, Progen, custom-made without any preservation components), anti-Dsg3 pAb (clone H-145, Santa Cruz Biotechnology, Santa Cruz, California), anti-Dsg3 mAb (clone 5G11, Life Technologies, Carlsbad, California), anti-Dsc1 pAb (clone L-15, Santa Cruz Biotechnology), anti-Dsc2 pAb (Progen), anti-Dsc3 mAb (clone U114, Progen), anti-ß-Actin mAb (Sigma, St.Louis, USA), anti-E-Cadherin mAb (clone 36, BD Biosciences), anti-Desmoplakin mAb (Epitomics, California, USA), anti-α-Tubulin mAb (Abcam, Cambridge, UK).

Techniques: Incubation, Microscopy, Binding Assay, Expressing, Immunofluorescence, Western Blot

Journal: PLoS ONE

Article Title: Desmoglein 2 Is Less Important than Desmoglein 3 for Keratinocyte Cohesion

doi: 10.1371/journal.pone.0053739

Figure Lengend Snippet: (A) Efficient Dsg2 (red) depletion was detected by immunofluorescence staining in HaCaT cells. Actin filaments are colored in green with Alexa Fluor®488 phalloidin. Scale bar, 20 µm. (B) Immunofluorescence staining for Dsg3 (red) and F-actin (green) after siRNA-mediated Dsg3 silencing. Scale bar, 20 µm. (C) Immunoblot analysis demonstrated a decrease in protein expression of either Dsg2 or Dsg3 after respective siRNA-mediated knockdown whereas protein levels of Dsc2 and E-cadherin were not changed after siRNA-mediated silencing of Dsg2 or Dsg3. Dsg2 depletion induced a slight but significant reduction of Dsg3 protein content. In contrast, Dsg2 levels were unchanged after Dsg3 silencing. ß-actin was used as loading control and band density was normalized to ß-actin. (n = 6; * p<0.05 vs. n. t. siRNA) (D) In contrast to Dsg3, endogenous Dsg2 expression was primarily detectable in the desmosomal pool (left panel). SiRNA-mediated gene silencing caused a reduction of Dsg3 in both protein fractions. Desmoplakin was used to identify the Triton X-100-insoluble fraction as the desmosome containing pool. Under control conditions, band density analysis revealed a five times higher ratio of insoluble over soluble protein levels of Dsg2 compared to Dsg3 indicating a more pronounced extradesmosomal localization of Dsg3 (right panel). (n = 5).

Article Snippet: Following primary antibodies were used to detect proteins by immunostaining and/or Western blot analysis: anti-Dsg1 (clone P124, Progen, Heidelberg, Germany), anti-Dsg2 mAb (clone 10G11, Progen, custom-made without any preservation components), anti-Dsg3 pAb (clone H-145, Santa Cruz Biotechnology, Santa Cruz, California), anti-Dsg3 mAb (clone 5G11, Life Technologies, Carlsbad, California), anti-Dsc1 pAb (clone L-15, Santa Cruz Biotechnology), anti-Dsc2 pAb (Progen), anti-Dsc3 mAb (clone U114, Progen), anti-ß-Actin mAb (Sigma, St.Louis, USA), anti-E-Cadherin mAb (clone 36, BD Biosciences), anti-Desmoplakin mAb (Epitomics, California, USA), anti-α-Tubulin mAb (Abcam, Cambridge, UK).

Techniques: Immunofluorescence, Staining, Western Blot, Expressing

Journal: PLoS ONE

Article Title: Desmoglein 2 Is Less Important than Desmoglein 3 for Keratinocyte Cohesion

doi: 10.1371/journal.pone.0053739

Figure Lengend Snippet: (A) Culture wells photographed after performance of the dispase-based dissociation assay under indicated conditions. Loss of cell-cell adhesion was detectable after AK23 incubation and siRNA-induced Dsg3 knockdown, but absent when Dsg2 levels were reduced by siRNA. (B) Mean fragment numbers per well under experimental conditions when control monolayers withstood the mechanical stress level and stayed intact. (n>20; * p<0.05 vs. n. t. siRNA) (C) Fragment numbers after exposing cell monolayers to higher mechanical stress. (n≥5; * p<0.05 vs. n. t. siRNA).

Article Snippet: Following primary antibodies were used to detect proteins by immunostaining and/or Western blot analysis: anti-Dsg1 (clone P124, Progen, Heidelberg, Germany), anti-Dsg2 mAb (clone 10G11, Progen, custom-made without any preservation components), anti-Dsg3 pAb (clone H-145, Santa Cruz Biotechnology, Santa Cruz, California), anti-Dsg3 mAb (clone 5G11, Life Technologies, Carlsbad, California), anti-Dsc1 pAb (clone L-15, Santa Cruz Biotechnology), anti-Dsc2 pAb (Progen), anti-Dsc3 mAb (clone U114, Progen), anti-ß-Actin mAb (Sigma, St.Louis, USA), anti-E-Cadherin mAb (clone 36, BD Biosciences), anti-Desmoplakin mAb (Epitomics, California, USA), anti-α-Tubulin mAb (Abcam, Cambridge, UK).

Techniques: Incubation

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: Frontiers in Immunology

Article Title: Role of Dsg1- and Dsg3-Mediated Signaling in Pemphigus Autoantibody-Induced Loss of Keratinocyte Cohesion

doi: 10.3389/fimmu.2019.01128

Figure Lengend Snippet: Genetic deletion of Dsg3 and Dsg2 using CRISPR/Cas9 in HaCaT keratinocytes. (A) Schematic of sequencing results after inducing a DSB with NHEJ repair in Exon 5 of either Dsg3 or Dsg2 using CRISPR/Cas9. (B) Cell line characterization by immunostaining of desmosomal proteins ( n = 3). (C) Cell line characterization by immunoblot with representative images on the left and densitometric quantification on the right ( n = 3, one-way ANOVA, * p ≤ 0.05 vs. WT). (D) Dispase assay of WT and Dsg-deficient cell lines under baseline treatment. ( n = 7, one-way ANOVA, * p ≤ 0.05 vs. WT) (E) Cell lines incubated with IgG fractions for 24 h and subjected to Dispase assays ( n = 5, two-way ANOVA, # p ≤ 0.05 vs. respective c-IgG, * p ≤ 0.05 vs. WT).

Article Snippet: The following primary antibodies were incubated overnight at 4°C: anti-Dsg3 mAb (clone 5G11, Invitrogen, Carlsbad, CA, USA), anti-Dsg2 mAb (OriGene, Herford, Germany), anti-E-Cadherin mAb (BD Transduction Laboratories, Heidelberg, Germany), anti-PG mAb (Progen, Heidelberg, Germany), anti-DP mAb (NW6, kind gift from Kathleen J.

Techniques: CRISPR, Sequencing, Immunostaining, Western Blot, Incubation

Journal: Frontiers in Immunology

Article Title: Role of Dsg1- and Dsg3-Mediated Signaling in Pemphigus Autoantibody-Induced Loss of Keratinocyte Cohesion

doi: 10.3389/fimmu.2019.01128

Figure Lengend Snippet: Signaling pathway modulation by pemphigus IgG fractions in Dsg-deficient cell lines. Analysis of ERK activation after 30 min application of IgG fractions by immunoblot in Dsg3- and Dsg2-deficient cell lines. (A) Representative immunoblot and (B) densitometric analysis ( n = 9, two-way ANOVA, * p ≤ 0.05). (C) Representativ graph of Fura-2 ratiometric Ca 2+ imaging reveals a PF-IgG mediated Ca 2+ influx in all cell lines used ( n = 3).

Article Snippet: The following primary antibodies were incubated overnight at 4°C: anti-Dsg3 mAb (clone 5G11, Invitrogen, Carlsbad, CA, USA), anti-Dsg2 mAb (OriGene, Herford, Germany), anti-E-Cadherin mAb (BD Transduction Laboratories, Heidelberg, Germany), anti-PG mAb (Progen, Heidelberg, Germany), anti-DP mAb (NW6, kind gift from Kathleen J.

Techniques: Activation Assay, Western Blot, Imaging