Journal: bioRxiv

Article Title: Defective Desmosomal Adhesion Causes Arrhythmogenic Cardiomyopathy by involving an Integrin-αVβ6/TGF-β Signaling Cascade

doi: 10.1101/2021.09.02.458734

Figure Lengend Snippet: ( A ) Venn diagram of significantly altered genes from indicated ACM patient data sets (ACM vs. healthy control) and DSG2-W2A mice at the age of 5 days and 9 weeks (mut/mut vs. wt/wt) highlighting integrin-β6 ( Itgb6 ) as only overlapping gene with same direction of expression in all data sets. Numbers indicate the amount of overlapping genes for the respective overlays. ( B ) RNA expression of Itgb6 analysed via q-RT-PCR in adult DSG2-W2A mouse hearts. *P< 0.05, unpaired Student’s t-test vs. wt/wt. ( C ) Representative Western blot and respective analysis of band intensity of ITGB6 in DSG2-W2A hearts. GAPDH served as loading control. *P< 0.05, one-way ANOVA, Dunnett’s post hoc test. ( D ) Immunostaining of ITGB6 (red in overlay) in DSG2-W2A hearts with corresponding analysis of staining intensity at membrane region in total and ratio of staining intensity at the lateral membrane (pink arrows) vs. ICD area (orange arrow heads). Desmoplakin (DSP, cyan) marks ICDs, DAPI (blue) nuclei and F-actin (green) the sarcomere system. Lower row shows an overview image of a fibrotic area in mut/mut hearts. Dotted orange line outlines the fibrotic area. Scale bars: upper rows: 20 µm, lower row: 50 µm. *P< 0.05, one-way ANOVA, Dunnett’s post hoc test. ( E ) Representative immunostainings of ITGB6 (red) and DSP (cyan) in an ACM patient (DSP-E952X) and a healthy control sample. Lateral membrane (pink arrows) and ICDs (orange arrow heads) are highlighted. F-actin (green) stains the sarcomere system. For the ACM patient, 4 different tissue samples were analysed and compared to 2 tissue samples from 2 healthy controls. Scale bar: 20 µm.

Article Snippet: The following primary antibodies were incubated in PBS at 4 °C overnight: Mouse anti-DSG1/2 (61002, Progen), mouse anti-DSP (61003, Progen), mouse anti-PG (61005, Progen), mouse anti-DSC2/3 (326200, Thermo Fisher Scientific), mouse anti-N-Cadherin (NCAD, 610921, BD Bioscience), rabbit anti-ITGB6 (ab187155, Abcam), rabbit anti-ITGB6/AV (BS-5791R, Bioss, Woburn, MA, USA), rabbit anti-ITGB1 (GTX112971, GeneTex, Irvine, CA, USA), rabbit anti-pSMAD2(S465/S467)/pSMAD3(S423/S425) (AP0548, Abclonal, Wuhan, China).

Techniques: Expressing, RNA Expression, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunostaining, Staining

Journal: bioRxiv

Article Title: Defective Desmosomal Adhesion Causes Arrhythmogenic Cardiomyopathy by involving an Integrin-αVβ6/TGF-β Signaling Cascade

doi: 10.1101/2021.09.02.458734

Figure Lengend Snippet: ( A ) Representative ICD reconstruction from z-stacks of ITGB6 immunostaining acquired by structured illumination microscopy (SIM). Lower row shows signal spot detection with indication of the average distance to the 5 nearest neighbour signals. Pictogram displays the angle of view. Related analysis of ITGB6 signal volume, number and average distance to the five nearest neighbours is shown below. Scale bar: 2 µm. *P< 0.05, unpaired Student’s t-test. Each dot represents the mean value of one ICD from in total 3 mice per genotype. ( B ) Representative images of ICDs acquired by transmission electron microscopy (TEM) with 3 mice per genotype. Orange asterisks mark intercellular widening, orange circle marks a ruptured junction. Scale bar: 1 µm. ( C ) Representative images of DSG2 (magenta) and filamentary actin (f-actin, white) stacks acquired by SIM and presented as z-stack maximum intensity projection. Lower row shows color-coded height projection of DSG2 signals in z-stack after signal thresholding as performed for analysis. Related analysis of DSG2 signal volume and number is shown below. Scale bar: 5 µm. *P< 0.05, unpaired Student’s t-test. Each dot represents the mean value of one ICD from in total 3 mice per genotype. ( D ) Representative immunoblot from immunoprecipitation (IP) of ITGB6 and DSG2 co-immunoprecipitation with analysis on the right. Intensity of co-immunoprecipitated DSG2 was normalized to the amount of pulled-down ITGB6. IP of IgG of the same species as anti-ITGB6 served as control for unspecific binding, M marks the lane with height marker. *P< 0.05, unpaired Student’s t-test. ( E ) Representative ICD reconstruction from z-stacks of ITGB6 (green) and DSG1/2 (magenta) immunostaining acquired by SIM. Analysis of the fraction of ITGB6 signals in close contact (< 200 nm) to a DSG2 signal and vice versa is shown on the right. Arrows highlight examples of colocalizing signals (white). Scale bar: 2 µm. *P< 0.05, unpaired Student’s t-test. Each dot represents the mean value of one ICD from in total 3 mice per genotype. ( F ) Immunostaining of ITGAV/B6 heterodimer in DSG2-W2A mutant hearts with respective analysis of staining intensity. Cyan rectangle marks zoomed area on the right. Scale bars: overview 50 µm; insert 10 µm. *P< 0.05, unpaired Student’s t-test. ( G ) Representative immunostaining images of ITGAV/B6 heterodimer staining (red) in a ACM patient (DSP-E952X) and healthy control sample. F-actin (green) stains the sarcomere system. For the ACM patient, 4 different tissue samples were analysed and compared to 2 tissue samples from 2 healthy controls. Scale bar: 20 µm.

Article Snippet: The following primary antibodies were incubated in PBS at 4 °C overnight: Mouse anti-DSG1/2 (61002, Progen), mouse anti-DSP (61003, Progen), mouse anti-PG (61005, Progen), mouse anti-DSC2/3 (326200, Thermo Fisher Scientific), mouse anti-N-Cadherin (NCAD, 610921, BD Bioscience), rabbit anti-ITGB6 (ab187155, Abcam), rabbit anti-ITGB6/AV (BS-5791R, Bioss, Woburn, MA, USA), rabbit anti-ITGB1 (GTX112971, GeneTex, Irvine, CA, USA), rabbit anti-pSMAD2(S465/S467)/pSMAD3(S423/S425) (AP0548, Abclonal, Wuhan, China).

Techniques: Immunostaining, Microscopy, Transmission Assay, Electron Microscopy, Western Blot, Immunoprecipitation, Binding Assay, Marker, Mutagenesis, Staining

Journal: bioRxiv

Article Title: Defective Desmosomal Adhesion Causes Arrhythmogenic Cardiomyopathy by involving an Integrin-αVβ6/TGF-β Signaling Cascade

doi: 10.1101/2021.09.02.458734

Figure Lengend Snippet: DSG2-W2A mutation with loss of desmosomal adhesion leads to impaired ICD structure with deregulation of ITGB6 and enhanced heterodimerization with ITGAV. The dimer efficiently binds to the extracellular matrix and activates TGF-β by removal of the latency-associated peptide (LAP). Active TGF-β can then induce pro-fibrotic downstream signaling via SMAD molecules. In our experiments, this cascade was blocked by anti-ITGAV/B6 or TGF-β receptor I inhibitor GW788388.

Article Snippet: The following primary antibodies were incubated in PBS at 4 °C overnight: Mouse anti-DSG1/2 (61002, Progen), mouse anti-DSP (61003, Progen), mouse anti-PG (61005, Progen), mouse anti-DSC2/3 (326200, Thermo Fisher Scientific), mouse anti-N-Cadherin (NCAD, 610921, BD Bioscience), rabbit anti-ITGB6 (ab187155, Abcam), rabbit anti-ITGB6/AV (BS-5791R, Bioss, Woburn, MA, USA), rabbit anti-ITGB1 (GTX112971, GeneTex, Irvine, CA, USA), rabbit anti-pSMAD2(S465/S467)/pSMAD3(S423/S425) (AP0548, Abclonal, Wuhan, China).

Techniques: Mutagenesis

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Integrin alpha-V is an important driver in pancreatic adenocarcinoma progression

doi: 10.1186/s13046-021-01946-2

Figure Lengend Snippet: Changes of ITGAV and corresponding beta-subunits ITGB1 und ITGB6 ( a ) using flow cytometry ( b ) and Western blot ( c ). The expression of ITGAV was reduced by > 65% in PaCa 5061 and BxPC3 ITGAV KD cells. Subunits ITGB1 and ITGB6 were found to be downregulated on PaCa 5061 ITGAV KD cells . In contrast, only ITGB6 was downregulated on BxPC3 ITGAV KD cells, while the expression of ITGB1 remained almost unchanged

Article Snippet: Following antibodies were used: ITGAV (327,907, BioLegend), ITGB1 (11–0299, Thermo Fisher Scientific), ITGB3 (336,403, BioLegend), ITGB5 (11–0497-42, Thermo Fisher Scientific), ITGB6 (FAB4155P, R&D), HLA-DR (347,401, BD).

Techniques: Flow Cytometry, Western Blot, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Integrin alpha-V is an important driver in pancreatic adenocarcinoma progression

doi: 10.1186/s13046-021-01946-2

Figure Lengend Snippet: Representative images of immunohistochemical staining of ITGAV, ITGB1, ITGB6, Ki-67, CEACAM7, TFBR1, pSMAD2, pSMAD2, TGFBI, Twist, E-cadherin, CD44, total and human fibronectin, STAT1 and HLA-DR ( a ) and quantification ( b ). The shown images are representative of three tumors harvested

Article Snippet: Following antibodies were used: ITGAV (327,907, BioLegend), ITGB1 (11–0299, Thermo Fisher Scientific), ITGB3 (336,403, BioLegend), ITGB5 (11–0497-42, Thermo Fisher Scientific), ITGB6 (FAB4155P, R&D), HLA-DR (347,401, BD).

Techniques: Immunohistochemical staining, Staining

Journal: Frontiers in Nuclear Medicine

Article Title: αvβ6-integrin targeted PET/CT imaging in pancreatic cancer patients using 68 Ga-Trivehexin

doi: 10.3389/fnume.2024.1487602

Figure Lengend Snippet: Examples for ITGB6 (β-6-integrin) immunohistochemistry (IHC) with corresponding PDAC-scores (a) PDAC-score 1; (b) PDAC-score 2; (c) PDAC-score 3.

Article Snippet: Human tissue was immunohistochemically examined using an anti-human Integrin β6 (ITGB6) mouse monoclonal antibody [clone 442.5C4] (#407317, dilution 1:100, Merck Millipore, Burlington, Massachusetts, USA).

Techniques: Immunohistochemistry