Journal: Cancer Management and Research

Article Title: DSG2 and c-MYC Interact to Regulate the Expression of ADAM17 and Promote the Development of Cervical Cancer

doi: 10.2147/CMAR.S456548

Figure Lengend Snippet: DSG2 is up-regulated in human cervical cancer and is associated with a poor prognosis. ( A ) Higher expression of DSG2 was found in cervical cancer samples than the normal tissues (based on TCGA database). ( B ) Kaplan–Meier plots of overall survival for cervical cancer samples with high/low DSG2 expression from the TCGA database.

Article Snippet: Primary antibodies against DSG2 (21,880-1-AP, 1:1000, Proteintech), ADAM17 (29,948-1-AP, 1:2000, Proteintech) and c-MYC (67,447-1-Ig, 1:5000, Proteintech) were used.

Techniques: Expressing

Journal: Cancer Management and Research

Article Title: DSG2 and c-MYC Interact to Regulate the Expression of ADAM17 and Promote the Development of Cervical Cancer

doi: 10.2147/CMAR.S456548

Figure Lengend Snippet: Knockdown of DSG2 in cervical cancer cells. ( A ) The knockdown efficiency of the three groups of siRNA in HeLa cells was 84%, 86% and 28%, respectively. ( B and C ) The specificity and validity of the siRNA knockdown of DSG2 expression in HeLa and SiHa cells was verified by qPCR ( B ) and WB ( C ). *P < 0.05, **P < 0.01.

Article Snippet: Primary antibodies against DSG2 (21,880-1-AP, 1:1000, Proteintech), ADAM17 (29,948-1-AP, 1:2000, Proteintech) and c-MYC (67,447-1-Ig, 1:5000, Proteintech) were used.

Techniques: Knockdown, Expressing

Journal: Cancer Management and Research

Article Title: DSG2 and c-MYC Interact to Regulate the Expression of ADAM17 and Promote the Development of Cervical Cancer

doi: 10.2147/CMAR.S456548

Figure Lengend Snippet: Knockdown of DSG2 inhibited proliferation and migration of cervical cancer cells. ( A ) The proliferation of HeLa and SiHa cells after knockdown of DSG2 was measured using CCK-8 assay. ( B ) The effect of DSG2 knockdown on cervical cancer cell clone formation. ( C ) The migration ability of HeLa and SiHa cells after knockdown of DSG2 was measured using Transwell assay. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Primary antibodies against DSG2 (21,880-1-AP, 1:1000, Proteintech), ADAM17 (29,948-1-AP, 1:2000, Proteintech) and c-MYC (67,447-1-Ig, 1:5000, Proteintech) were used.

Techniques: Knockdown, Migration, CCK-8 Assay, Transwell Assay

Journal: Cancer Management and Research

Article Title: DSG2 and c-MYC Interact to Regulate the Expression of ADAM17 and Promote the Development of Cervical Cancer

doi: 10.2147/CMAR.S456548

Figure Lengend Snippet: ADAM17 is up-regulated in human cervical cancer and is associated with a poor prognosis. ( A ) The bioinformatics analysis of TCGA-CHOL dataset showed the positive expression correlation between DSG2 and ADAM17. ( B ) Higher expression of ADAM17 was found in cervical cancer samples than the normal tissues (based on TCGA database). ( C ) Kaplan–Meier plots of overall survival for cervical cancer samples with high/low ADAM17 expression from the TCGA database.

Article Snippet: Primary antibodies against DSG2 (21,880-1-AP, 1:1000, Proteintech), ADAM17 (29,948-1-AP, 1:2000, Proteintech) and c-MYC (67,447-1-Ig, 1:5000, Proteintech) were used.

Techniques: Expressing

Journal: Cancer Management and Research

Article Title: DSG2 and c-MYC Interact to Regulate the Expression of ADAM17 and Promote the Development of Cervical Cancer

doi: 10.2147/CMAR.S456548

Figure Lengend Snippet: DSG2 regulates the expression of ADAM17 in cervical cancer. ( A ) The effect of DSG2 knockdown on ADAM17mRNA expression was detected by qPCR. ( B ) The effect of DSG2 knockdown on ADAM17 protein expression was detected by WB. ( C ) The interaction between DSG2 and c-MYC was detected by Co-IP assay. ***P < 0.001.

Article Snippet: Primary antibodies against DSG2 (21,880-1-AP, 1:1000, Proteintech), ADAM17 (29,948-1-AP, 1:2000, Proteintech) and c-MYC (67,447-1-Ig, 1:5000, Proteintech) were used.

Techniques: Expressing, Knockdown, Co-Immunoprecipitation Assay

Journal: Cancer Management and Research

Article Title: DSG2 and c-MYC Interact to Regulate the Expression of ADAM17 and Promote the Development of Cervical Cancer

doi: 10.2147/CMAR.S456548

Figure Lengend Snippet: DSG2 regulates cervical cancer development by interacting with c-MYC. ( A ) HeLa cells were transfected with pcDNA(3.1)-DSG2 overexpression plasmid, and the mRNA level of DSG2 was detected by qPCR. ( B ) HeLa cells were transfected with pcDNA(3.1) overexpression plasmid, and the protein level of DSG2 was detected by WB. ( C and D ) DSG2 overexpressed HeLa cells were treated with a C-MYC inhibitor (10,058-F4, 50 μM), and the proliferative activity and migration ability were detected by CCK-8 ( C ) and clonal formation assay ( D ). ( E ) DSG2 overexpressed HeLa cells were treated with a C-MYC inhibitor, and ADAM17 protein expression was detected by WB.*P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Primary antibodies against DSG2 (21,880-1-AP, 1:1000, Proteintech), ADAM17 (29,948-1-AP, 1:2000, Proteintech) and c-MYC (67,447-1-Ig, 1:5000, Proteintech) were used.

Techniques: Transfection, Over Expression, Plasmid Preparation, Activity Assay, Migration, CCK-8 Assay, Tube Formation Assay, Expressing

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: Samples (50 lg per lane) were resolved in 4– 12% Bis-Tris polyacrylamide gels (Bio-Rad, Hercules, CA, USA) and electrophoretically transferred to nitrocellulose filters (Pall Corporation, New York, NY, USA) prior to blocking (Odyssey Blocking Buffer, LiCOR, Lincoln, NE, USA) and incubation with antibodies to DSG2 (Bethyl Laboratories, Montgomery, TX, USA), phospho-p44/42 MAPK (ERK1/2, Thr202/ Tyr204; CST), phospho-AKT (Ser473; CST) or IƘBa (Cell Signaling Technology) at 1 : 1000 dilution in blocking buffer for 1 h. Washed filters were incubated for 45 min with either IRDye 800CW goat anti-rabbit IgG (Li-COR) or IRDye 680CW goat anti-mouse at a 1 : 10 000 dilution in blocking buffer.

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: Samples (50 lg per lane) were resolved in 4– 12% Bis-Tris polyacrylamide gels (Bio-Rad, Hercules, CA, USA) and electrophoretically transferred to nitrocellulose filters (Pall Corporation, New York, NY, USA) prior to blocking (Odyssey Blocking Buffer, LiCOR, Lincoln, NE, USA) and incubation with antibodies to DSG2 (Bethyl Laboratories, Montgomery, TX, USA), phospho-p44/42 MAPK (ERK1/2, Thr202/ Tyr204; CST), phospho-AKT (Ser473; CST) or IƘBa (Cell Signaling Technology) at 1 : 1000 dilution in blocking buffer for 1 h. Washed filters were incubated for 45 min with either IRDye 800CW goat anti-rabbit IgG (Li-COR) or IRDye 680CW goat anti-mouse at a 1 : 10 000 dilution in blocking buffer.

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: Samples (50 lg per lane) were resolved in 4– 12% Bis-Tris polyacrylamide gels (Bio-Rad, Hercules, CA, USA) and electrophoretically transferred to nitrocellulose filters (Pall Corporation, New York, NY, USA) prior to blocking (Odyssey Blocking Buffer, LiCOR, Lincoln, NE, USA) and incubation with antibodies to DSG2 (Bethyl Laboratories, Montgomery, TX, USA), phospho-p44/42 MAPK (ERK1/2, Thr202/ Tyr204; CST), phospho-AKT (Ser473; CST) or IƘBa (Cell Signaling Technology) at 1 : 1000 dilution in blocking buffer for 1 h. Washed filters were incubated for 45 min with either IRDye 800CW goat anti-rabbit IgG (Li-COR) or IRDye 680CW goat anti-mouse at a 1 : 10 000 dilution in blocking buffer.

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: Samples (50 lg per lane) were resolved in 4– 12% Bis-Tris polyacrylamide gels (Bio-Rad, Hercules, CA, USA) and electrophoretically transferred to nitrocellulose filters (Pall Corporation, New York, NY, USA) prior to blocking (Odyssey Blocking Buffer, LiCOR, Lincoln, NE, USA) and incubation with antibodies to DSG2 (Bethyl Laboratories, Montgomery, TX, USA), phospho-p44/42 MAPK (ERK1/2, Thr202/ Tyr204; CST), phospho-AKT (Ser473; CST) or IƘBa (Cell Signaling Technology) at 1 : 1000 dilution in blocking buffer for 1 h. Washed filters were incubated for 45 min with either IRDye 800CW goat anti-rabbit IgG (Li-COR) or IRDye 680CW goat anti-mouse at a 1 : 10 000 dilution in blocking buffer.

Techniques: Gene Expression, Expressing

Journal: Journal of Virology

Article Title: Impact of Human Adenovirus Type 3 Dodecahedron on Host Cells and Its Potential Role in Viral Infection

doi: 10.1128/JVI.07127-11

Figure Lengend Snippet: Escape of penton dodecahedron (Pt-Dd) from Ad3-infected cells. (A) HeLa cells were infected with 0.1 PFU/cell wild-type Ad3 for 24, 48, or 72 h, fixed with 10% formalin, and permeabilized with 0.1% Triton X-100. Cells were stained for DSG-2 expression using rabbit polyclonal anti-DSG-2 (GeneTex) and fluorescein isothiocyanate (FITC)-labeled anti-rabbit secondary antibodies (Jackson ImmunoResearch). Dodecahedra were localized using mouse monoclonal anti-fiber antibodies (Abcam, United Kingdom) and Cy3-labeled anti-mouse secondary antibodies (Jackson ImmunoResearch). Cell nuclei were stained using DAPI (4′,6-diamidino-2-phenylindole) (in blue). A close-up view at 48 hpi (white rectangle) shows orange dots along the DSG-2 signal at the contact between two cells. Infected cells are indicated by white stars. n.i., not infected. (B) HeLa cells were infected with wild-type Ad3, fixed, and permeabilized as described above. Dodecahedra were then identified using rabbit polyclonal anti-Pt-Dd (2) and FITC-labeled anti-rabbit secondary antibodies. Pt-Dd staining in noninfected cells is denoted by white arrows. In the lower panel, Pt-Dd staining was observed at the interface between two adjacent cells. Cells were observed using an Olympus IX81 microscope.

Article Snippet: Cells were fixed, permeabilized, and incubated for 1 h at room temperature with a 1:250 dilution of rabbit anti-DSG-2 antibody (GeneTex Inc.).

Techniques: Infection, Staining, Expressing, Labeling, Microscopy

Journal: Journal of Virology

Article Title: Impact of Human Adenovirus Type 3 Dodecahedron on Host Cells and Its Potential Role in Viral Infection

doi: 10.1128/JVI.07127-11

Figure Lengend Snippet: Pt-Dd interactions with the Ad3 attachment molecule desmoglein-2. (A) To assess competition between dodecahedra and Ad3-EGFP, Bs-Dd or Pt-Dd was added to A549 cells for 30 min at room temperature prior to transduction with replication-deficient Ad3-EGFP at 1 fluorescent forming unit (FFU)/cell. Twenty-four hours later, EGFP expression was analyzed by flow cytometry. Relative geometric means are derived from EGFP expression in the absence of Dd (100%). White bars represent data from Bs-Dd-treated cells, and black bars represent data from Pt-Dd-treated cells. (B) Dodecahedra (1 μg) were added to A549 cells and incubated at 37°C for 2 h. Cells were then washed twice with phosphate-buffered saline (PBS), and Ad3-EGFP was added in serum-free Dulbecco's modified Eagle medium (DMEM) at 1 FFU/cell. Following an additional 2-h incubation at 37°C, cells were washed twice using PBS, and 2 ml of DMEM supplemented with 10% fetal bovine serum (FBS) was added. Cells were incubated overnight at 37°C, and EGFP expression was determined by flow cytometry. (C) To monitor DSG-2 internalization with Pt-Dd, A549 cells were incubated at 37°C for 2 h with Cy5–Pt-Dd (red signal) in DMEM. Cells were fixed, permeabilized, and incubated for 1 h at room temperature with a 1:250 dilution of rabbit anti-DSG-2 antibody (GeneTex Inc.). After PBS washes, cells were incubated for 30 min at room temperature with a 1:250 dilution of FITC anti-rabbit immunoglobulins (Jackson ImmunoResearch) (green signal). DIC, differential interference contrast. (D) Dodecahedra were added to primary human endothelial cells (HUVEC) for 30 min at room temperature prior to transduction with Ad3-EGFP (1 FFU/cell). Twenty-four hours later, EGFP expression was detected by flow cytometry. Relative geometric means are derived from EGFP expression in the absence of Dd (100%). White bars represent data from Bs-Dd-treated cells, and black bars represent data from Pt-Dd-treated cells.

Article Snippet: Cells were fixed, permeabilized, and incubated for 1 h at room temperature with a 1:250 dilution of rabbit anti-DSG-2 antibody (GeneTex Inc.).

Techniques: Transduction, Expressing, Flow Cytometry, Derivative Assay, Incubation, Modification

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, Gene Expression, RNA Sequencing

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, Gene Expression, 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, Gene Expression, Knockdown, Flow Cytometry, Staining, Immunofluorescence, Positive Control, Negative Control

Journal: PloS one

Article Title: In vitro functional analyses of arrhythmogenic right ventricular cardiomyopathy-associated desmoglein-2-missense variations.

doi: 10.1371/journal.pone.0047097

Figure Lengend Snippet: Figure 1. A Schematic view of the rECD with analysed ARVC-associated variations. The dotted line shows the predicted PC cleavage site. SS = signal sequence, Pro = prodomain, EC1-EC4 = DSG2 extracellular cadherin subdomains 1-4. B Recombinantly expressed proteins were identified as DSG2-ECD with anti-DSG2-10G11 by Western blot analysis. The calculated apparent molecular weights were 67.561.5, 72.563.5, 70.063.0, 70.063.0, 70.562.5, and 69.064.0 (mean6SEM; n = 2) for the proteins in the traces in 1, 2, 3, 4, 5 and 6, respectively. C Coomassie-R-250 staining revealed the purity of the proteins. 1 = rECD-wt, 2-6 = rECDs as labelled in A. doi:10.1371/journal.pone.0047097.g001

Article Snippet: For Western blotting a murine IgG1 antibody against the extracellular domain of DSG2 clone 10G11 (anti-DSG2-10G11; BM5016, Acris Antibodies GmbH), a murine IgG1 antibody clone DG3.10 against the intracellular domain of DSG1+2 (antiDSG1+2-DG3.10; BM370, Acris Antibodies GmbH) and a rabbit polyclonal antibody to PDI IgG (anti-PDI; ab31811, Abcam) as an endoplasmatic reticulum marker were used.

Techniques: Sequencing, Western Blot, Staining

Journal: PloS one

Article Title: In vitro functional analyses of arrhythmogenic right ventricular cardiomyopathy-associated desmoglein-2-missense variations.

doi: 10.1371/journal.pone.0047097

Figure Lengend Snippet: Figure 5. Adhesion properties of rECDs. A+B Flow cytometry- based assay for the binding of 0.8 mM rECD-wt or -variants to HT1080. A Representative histograms of FITC- fluorescence for binding of rECD-wt- and rECD-R46Q (as indicated). Bound rECD was detected with anti- HisFITC. As a negative control, HT1080 cells were incubated with only anti-HisFITC (grey filled area). B Column plots representing the ratio of rECD-binding related to the negative control (ratiorECD-bound) as detected by flow cytometry. RatiosrECD-bound are indicated as mean6 SEM of 7 independent measurements for rECD-variants and 9 independent measurements for rECD-wt with rECDs from at least 3 different purifications. Statistical analysis was performed by one-way ANOVA with Dunnett’s posttest using rECD-wt as a control (GraphPad Prism 5.01). rECD-R46Q-binding to HT1080 is increased 1.8-fold as compared to rECD-wt. Other ARVC-associated variants have no influence on rECD-binding to HT1080. C Representative Western blot (with anti-DSG2-10G11) of rECDs crosslinked in a 5 mM CaCl2 contain- ing buffer with BS3 (+) or of controls (-) reveals that rECD wild-type and variants exist in solution as monomers (m), dimers (d), and oligo- mers (o). doi:10.1371/journal.pone.0047097.g005

Article Snippet: For Western blotting a murine IgG1 antibody against the extracellular domain of DSG2 clone 10G11 (anti-DSG2-10G11; BM5016, Acris Antibodies GmbH), a murine IgG1 antibody clone DG3.10 against the intracellular domain of DSG1+2 (antiDSG1+2-DG3.10; BM370, Acris Antibodies GmbH) and a rabbit polyclonal antibody to PDI IgG (anti-PDI; ab31811, Abcam) as an endoplasmatic reticulum marker were used.

Techniques: Flow Cytometry, Binding Assay, Fluorescence, Negative Control, Incubation, Control, 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: 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

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