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: Cell & Bioscience

Article Title: Protein expression of eIF4E and integrin αvβ6 in colon cancer can predict clinical significance, reveal their correlation and imply possible mechanism of interaction

doi: 10.1186/2045-3701-4-23

Figure Lengend Snippet: Association between eIF4E expression, integrin αvβ6 expression and clinicopathologic variables in colon cancer cases

Article Snippet: As primary antibodies, prediluted anti-Integrin β6 polyclonal rabbit antibody (1:200; Proteintech Group, Inc., Cat.No.19695-1-AP, Chicago, USA), anti- eIF-4E rabbit monoclonal antibody (1:100; Epitomics, Inc., Cat.No.

Techniques: Expressing

Journal: Cell & Bioscience

Article Title: Protein expression of eIF4E and integrin αvβ6 in colon cancer can predict clinical significance, reveal their correlation and imply possible mechanism of interaction

doi: 10.1186/2045-3701-4-23

Figure Lengend Snippet: Integrin β6 expression in human colonic cancer. First row: (a) , (b) , (c) (Scale bar 200 μm.); second row: (d) , (e) , (f) , corresponding to each figure above (Scale bar 100 μm). (a) and (d) was obtained from paracancerous normal tissue of patient with colon cancer, with negative staining. (b) and (e) was obtained from colonic carcinoma tissue,with negative Integrin β6 expression. (c) and (f) was obtained from colonic carcinoma, with positive Integrin β6 expression.

Article Snippet: As primary antibodies, prediluted anti-Integrin β6 polyclonal rabbit antibody (1:200; Proteintech Group, Inc., Cat.No.19695-1-AP, Chicago, USA), anti- eIF-4E rabbit monoclonal antibody (1:100; Epitomics, Inc., Cat.No.

Techniques: Expressing, Negative Staining

Journal: Cell & Bioscience

Article Title: Protein expression of eIF4E and integrin αvβ6 in colon cancer can predict clinical significance, reveal their correlation and imply possible mechanism of interaction

doi: 10.1186/2045-3701-4-23

Figure Lengend Snippet: Correlation between integrin αvβ6 expression and eIF4E expression in human colonic carcinoma tissues (r = 0.299, P < 0.001)

Article Snippet: As primary antibodies, prediluted anti-Integrin β6 polyclonal rabbit antibody (1:200; Proteintech Group, Inc., Cat.No.19695-1-AP, Chicago, USA), anti- eIF-4E rabbit monoclonal antibody (1:100; Epitomics, Inc., Cat.No.

Techniques: Expressing

Journal: Cell & Bioscience

Article Title: Protein expression of eIF4E and integrin αvβ6 in colon cancer can predict clinical significance, reveal their correlation and imply possible mechanism of interaction

doi: 10.1186/2045-3701-4-23

Figure Lengend Snippet: Overall survival according to eIF4E with integrin αvβ6 expression (P = 0.028, The log-rank test).

Article Snippet: As primary antibodies, prediluted anti-Integrin β6 polyclonal rabbit antibody (1:200; Proteintech Group, Inc., Cat.No.19695-1-AP, Chicago, USA), anti- eIF-4E rabbit monoclonal antibody (1:100; Epitomics, Inc., Cat.No.

Techniques: Expressing

Journal: Annals of Translational Medicine

Article Title: Silencing of ITGB6 inhibits the progression of cervical carcinoma via regulating JAK/STAT3 signaling pathway

doi: 10.21037/atm-21-1669

Figure Lengend Snippet: ITGB6 was upregulated in CC. (A,B) TCGA data sets showed ITGB6 expression was higher in tumor tissues than in normal tissues; (C) Kaplan-Meier survival plots demonstrated that higher ITGB6 abundance correlated with a poorer overall survival according to TCGA database; (D,E) The expression of ITGB6 in 25 CC tumor tissues and normal tissues was examined using qRT-PCR and IHC (magnification: 100×), respectively. **P<0.01, compared with NT. ITGB6, Integrin β6; CC, cervical carcinoma; TCGA, The Cancer Genome Atlas; N0, node negative; N1, node positive (1–3 positive nodes); NT, normal tissues; qRT-PCR, quantitative real-time polymerase chain reaction; IHC, immunohistochemistry.

Article Snippet: Subsequently, the slides were incubated with a rabbit polyclonal anti-ITGB6 antibody (ab233519, 1:100, Abcam, USA) overnight at 4 °C.

Techniques: Expressing, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Immunohistochemistry

Journal: Annals of Translational Medicine

Article Title: Silencing of ITGB6 inhibits the progression of cervical carcinoma via regulating JAK/STAT3 signaling pathway

doi: 10.21037/atm-21-1669

Figure Lengend Snippet: Silencing of ITGB6 suppressed cell proliferation and promoted apoptosis in CC cells. (A) The ITGB6 expression in CC cells lines (SiHa, Caski C-33A, and Hela) and human cervical immortalized squamous cell line (ECT1/E6E7) was tested by qRT-PCR; (B) relative mRNA expression levels of ITGB6 in SiHa and Hela cells transfected with ITGB6 siRNAs (si1-ITGB6 and si2-ITGB6) or NC siRNAs (si-NC); (C,D,E) the cell viability, proliferative capability, and cell apoptosis of transfected SiHa and Hela cells was evaluated by CKK-8, colon-formation (crystal violet stain, magnification: 40×), and flow cytometry assay, respectively. **P<0.01, compared with ECT1/E6E7, or the si-NC group. ITGB6, Integrin β6; CC, cervical carcinoma; qRT-PCR, quantitative real-time polymerase chain reaction; si-NC, negative control siRNAs; CKK-8, Cell Counting Kit-8; OD, optical density.

Article Snippet: Subsequently, the slides were incubated with a rabbit polyclonal anti-ITGB6 antibody (ab233519, 1:100, Abcam, USA) overnight at 4 °C.

Techniques: Expressing, Quantitative RT-PCR, Transfection, Staining, Flow Cytometry, Real-time Polymerase Chain Reaction, Negative Control, Cell Counting

Journal: Annals of Translational Medicine

Article Title: Silencing of ITGB6 inhibits the progression of cervical carcinoma via regulating JAK/STAT3 signaling pathway

doi: 10.21037/atm-21-1669

Figure Lengend Snippet: Silencing of ITGB6 inhibited the invasion, migration, and EMT of CC cells. (A,B) SiHa and Hela cell migration and invasion was tested by Transwell assays (crystal violet stain, magnification: 40×). (C) Western blotting assay was used to evaluate the protein expression levels of Snail, vimentin, N-cadherin, and E-cadherin in SiHa and Hela. **P<0.01, compared with the si-NC group. ITGB6, Integrin β6; CC, cervical carcinoma; EMT, epithelial-to-mesenchymal transition; si-NC, negative control siRNAs.

Article Snippet: Subsequently, the slides were incubated with a rabbit polyclonal anti-ITGB6 antibody (ab233519, 1:100, Abcam, USA) overnight at 4 °C.

Techniques: Migration, Staining, Western Blot, Expressing, Negative Control

Journal: Annals of Translational Medicine

Article Title: Silencing of ITGB6 inhibits the progression of cervical carcinoma via regulating JAK/STAT3 signaling pathway

doi: 10.21037/atm-21-1669

Figure Lengend Snippet: Silencing of ITGB6 inhibited the JAK/STAT3 signaling pathway in CC cells. (A) GSEA analysis found that ITGB6 expression was positively correlated with HALLMARK IL6/JAK/STAT3 signaling pathway; (B) Western blotting assay was applied to detect the JAK/STAT3 signaling pathway-related proteins (JAK1, p-JAK1, JAK2, p-JAK2, p-STAT3, and STAT3) in SiHa and Hela cells. **P<0.01, compared with the si-NC group. ITGB6, Integrin β6; CC, cervical carcinoma; GSEA, gene set enrichment analysis; IL6/JAK/STAT3, Interleukin-6/janus kinase/signal transducer and activator of transcription; si-NC, negative control siRNAs.

Article Snippet: Subsequently, the slides were incubated with a rabbit polyclonal anti-ITGB6 antibody (ab233519, 1:100, Abcam, USA) overnight at 4 °C.

Techniques: Expressing, Western Blot, Negative Control

Journal: Annals of Translational Medicine

Article Title: Silencing of ITGB6 inhibits the progression of cervical carcinoma via regulating JAK/STAT3 signaling pathway

doi: 10.21037/atm-21-1669

Figure Lengend Snippet: Silencing of ITGB6 suppressed CC progression via regulating JAK-STAT3 signaling. (A) Western blotting assay was applied to detect the JAK/STAT3 signaling pathway-related proteins (JAK1, p-JAK1, JAK2, p-JAK2, p-STAT3, and STAT3) in Hela and SiHa cells; (B) The cell proliferation of Hela and SiHa cells was evaluated by colony formation (crystal violet stain, magnification: 40×); (C,D) Transwell assay was used to evaluate the migration and invasion of Hela and SiHa cells (crystal violet stain, magnification: 40×); (E) Western blotting assay was applied to detect the EMT-related proteins (Snail, vimentin, N-cadherin, and E-cadherin) in Hela and SiHa cells. **P<0.01, compared with si-NC; ##P<0.01, compared with the si1-ITGB6 group, &&P<0.01, compared with the si-NC + RO8191 group. ITGB6, Integrin β6; CC, cervical carcinoma; JAK/STAT3, Janus kinase/signal transducer and activator of transcription; EMT, epithelial-to-mesenchymal transition; si-NC, negative control siRNAs.

Article Snippet: Subsequently, the slides were incubated with a rabbit polyclonal anti-ITGB6 antibody (ab233519, 1:100, Abcam, USA) overnight at 4 °C.

Techniques: Western Blot, Staining, Transwell Assay, Migration, Negative Control

Journal: Clinical and Translational Gastroenterology

Article Title: β 6 -Integrin Serves as a Potential Serum Marker for Diagnosis and Prognosis of Pancreatic Adenocarcinoma

doi: 10.14309/ctg.0000000000000395

Figure Lengend Snippet: Validation of ITGB6 as possible biomarker for PAC. ( a ) Serum ITGB6 levels were assessed in a prospective study cohort of 27 patients with PAC (cohort 1, N = 27). As control served 9 healthy volunteers (Ctrl; N = 9) and 10 patients with cP (N = 10). Significant differences in ITGB6 levels were observed between patients with PAC and Ctrl ( P = 0.019). ( b ) Comparing patients with or without distant metastatic PAC, serum ITGB6 levels were not significantly different. However, a significant increase in ITGB6 levels was observed between Ctrl and patients with nonmetastatic PAC ( P = 0.019). ( c ) To assess the prognostic value of serum ITGB6 levels, patients with PAC were plotted against their status of survival at time of blood assessment. A significant difference in ITGB6 concentration was observed between patients with PAC with status alive vs dead ( P = 0.007). ( d ) Two-dimensional scatterplots depict serum ITGB6 levels in relation to the serum CA19-9 levels from each cP ( r s = 0.511; P = 0.132) and patient with PAC ( r s = 0.210; P = 0.302). In ( a – c ), Mann–Whitney U Exact and Sig. 2-Tailed test were performed, and in ( d ), Spearman's rho correlation ( r s ) and Sig. 2-tailed test were performed. ( d ) Red lines indicate ITGB6 cutoff value at 0.1 ng/mL and black line CA19-9 cutoff value at 37.0 kU/L, respectively. cP, chronic pancreatitis; Ctrl, control; ITGB6, β 6 -integrin; PAC, pancreatic adenocarcinoma; r s, Spearman's rho correlation. *Means P < 0.05.

Article Snippet: A tissue microarray (TMA) with formalin-fixed paraffin-embedded PAC tissue specimens from 83 patients was automatically stained with a human anti-ITGB6 polyclonal antibody raised in rabbit (HPA023626, dilution 1:100; Atlas Antibodies AB, Bromma, Sweden) using a validated standard protocol on a Ventana automat (Ventana Medical System, Tucson, AZ).

Techniques: Biomarker Discovery, Control, Concentration Assay, MANN-WHITNEY

Journal: Clinical and Translational Gastroenterology

Article Title: β 6 -Integrin Serves as a Potential Serum Marker for Diagnosis and Prognosis of Pancreatic Adenocarcinoma

doi: 10.14309/ctg.0000000000000395

Figure Lengend Snippet: Serum ITGB6 levels may predict overall and progression-free survival in patients with PAC. ( a ) Survival rates and ( b ) progression-free survival of patients with PAC who underwent systemic therapy are plotted according to serum ITGB6 changes after initiation (N = 24; cohort 2). ( c ) Two-dimensional scatterplots depict lymph node ratio (LNR) in relation to exits after surgery (months) for each patient in accordance to the ITGB6 protein expression pattern. Hazard ratio (HR) was calculated with the Cox regression. ( d ) Overall survival in AU12 patients with PAC (N582; cohort 3) was plotted according to ITGB6 protein expression pattern. Kaplan-Meier estimation was performed and plotted accordingly. The log-rank P values are indicated. ITGB6, β 6 -integrin; PAC, pancreatic adenocarcinoma.

Article Snippet: A tissue microarray (TMA) with formalin-fixed paraffin-embedded PAC tissue specimens from 83 patients was automatically stained with a human anti-ITGB6 polyclonal antibody raised in rabbit (HPA023626, dilution 1:100; Atlas Antibodies AB, Bromma, Sweden) using a validated standard protocol on a Ventana automat (Ventana Medical System, Tucson, AZ).

Techniques: Expressing

Journal: Clinical and Translational Gastroenterology

Article Title: β 6 -Integrin Serves as a Potential Serum Marker for Diagnosis and Prognosis of Pancreatic Adenocarcinoma

doi: 10.14309/ctg.0000000000000395

Figure Lengend Snippet: ITGB6 protein expression in PAC tissue specimens by IHC. Representative TMA specimens (from cohort 3, N = 83) illustrate ITGB6-negative (no [0] or mild [1+] expression) and ITGB6-positive PAC specimens (moderate [2+] or strong [3+] expression; scale bars: upper panel 200 μm; lower panel 100 μm). IHC, immunohistochemistry; ITGB6, β 6 -integrin; PAC, pancreatic adenocarcinoma; TMA, tissue microarray.

Article Snippet: A tissue microarray (TMA) with formalin-fixed paraffin-embedded PAC tissue specimens from 83 patients was automatically stained with a human anti-ITGB6 polyclonal antibody raised in rabbit (HPA023626, dilution 1:100; Atlas Antibodies AB, Bromma, Sweden) using a validated standard protocol on a Ventana automat (Ventana Medical System, Tucson, AZ).

Techniques: Expressing, Immunohistochemistry, Microarray

Journal: Cell Death & Disease

Article Title: Myofibroblast induces hepatocyte-to-ductal metaplasia via laminin–ɑvβ6 integrin in liver fibrosis

doi: 10.1038/s41419-020-2372-9

Figure Lengend Snippet: Key resourse table.

Article Snippet: Rabbit anti-ITGB6 , ABclonal technology , WB , A16904.

Techniques: Immunohistochemistry-IF, Flow Cytometry, Recombinant, Solvent, Cell Culture, Staining, RNA Extraction, Isolation, Transfection, Software, Imaging