human β1 integrin  (Developmental Studies Hybridoma Bank)

Journal: Nature Communications

Article Title: Galectin-3-integrin α5β1 phase separation disrupted by advanced glycation end-products impairs diabetic wound healing in rodents

doi: 10.1038/s41467-025-62320-w

Figure Lengend Snippet: A Quantifications of HMEC-1 cell migration (left) and tube formation (right) induced by recombinant Gal-3 under an insulin-resistant state (treated with serum-free medium containing 100 nM insulin for 24 h) were shown ( n = 4 biological replicates each group). -, normal state; +, insulin-resistant state. B RT-qPCR analysis of VEGFA , FGF2 , and HGF in HMEC-1 cells treated with the indicated concentration of recombinant Gal-3 for 12 h. Relative expression levels were normalized to ACTB ( n = 3 biological replicates each group). C Heatmap of proteomic abundance (normalized using Z-score) of the top 5 Gal-3-interacting proteins in skin endothelial cells from healthy donors (dataset PXD019909, ProteomeXchange). D HMEC-1 cell migration (left) ( n = 4 biological replicates) and tube formation (right) ( n = 3 biological replicates) induced by recombinant Gal-3 with knockdown of Catenin α-1 or integrin β1. E GST pull-down assays. HMEC-1 cell lysate was incubated with GST or GST-Gal-3 and pulled down with GS beads (left panel); cells were treated with GST or GST-Gal-3 at 4 °C for 1 h, cross-linked, lysed and pulled down with GS beads (right panel). GST served as a negative control. Immunoblot analysis of integrin β1 was shown. F Recombinant Gal-3-induced migration of HMEC-1 cells with integrin β1-targeting shRNAs or non-targeting shRNA (shscr) ( n = 3 biological replicates each group). G Recombinant Gal-3-induced migration of HMEC-1 cells incubated with integrin β1 functional blocking antibody (TDM29, 10 µg/mL) or IgG control (left). Quantifications were shown ( n = 3 biological replicates each group). Scale bar, 500 μm. H Schematic diagram of the α subunit partnering with the integrin β1 subunit created in BioRender. Chen, S. (2025) https://BioRender.com/p10vue6 . Among the 12 α subunits, α3, α5, and α6 subunits were detected by the mass spectrometry analysis in the His-Gal-3 immunoprecipitation assay performed in HMEC-1 cells (see Supplementary Fig. ). I GST pull-down assays. HMEC-1 cell lysate was incubated with GST or GST-Gal-3 and pulled down with GS beads (left panel); cells were treated with GST or GST-Gal-3 at 4 °C for 1 h, cross-linked, lysed and pulled down with GS beads (right panel). GST served as a negative control. Immunoblot analysis of integrin α5, integrin α6 and integrin α3 was shown. J Recombinant Gal-3 induced migration of HMEC-1 cells incubated with integrin α5 functional blocking antibody P1D6 (10 µg/mL) ( n = 3 biological replicates each group). K Recombinant Gal-3-induced migration of HMEC-1 cells that were pre-incubated with integrin α5β1 antagonist ATN-161 (100 nM) for 48 h ( n = 5 biological replicates each group). L , Immunoblot analysis of the phosphorylation of integrin β1 (p-integrin β1, Ser783) in HMEC-1 cells that were incubated with recombinant Gal-3 (10 μg/mL). Relative expression levels were normalized to integrin β1, and quantifications were shown below the blots. M Immunoblot analysis and quantifications of p-integrin β1 in wounds of diabetic mice that i.c . injected with OE-Gal-3 adenovirus or Veh. Relative expression levels were normalized to integrin β1 ( n = 3 biological replicates each group). All statistical data points are represented as means ± SEM. P values were determined by unpaired two-tailed Student’s t -test ( A , B , D , F , G , J , K , M ) or one-way ANOVA with Fisher’s LSD post hoc test ( B , D , F ). Error bars mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001. Source data are provided as a Source Data file. Exact p values are provided in the Source Data file.

Article Snippet: Purified GFP-Gal-3 (80 μM) was mixed with integrin β1 (400 nM) (10587-H08H1, Sino Biological, China), integrin α5β1 (400 nM), integrin β1 (400 nM) + lactose (20 mM), integrin α5β1 (400 nM) + lactose (20 mM), or CD146 (400 nM) in PBS and incubated at 37 °C for 5 min. PNGase F-treated integrin α5β1 was prepared by incubating 4 μg of integrin α5β1 with 1000 U of PNGase F at 37 °C for 2 h using a PNGase F Kit (P0704S, NEW ENGLAND BioLabs, China) according to the manufacturer’s instructions.

Techniques: Migration, Recombinant, Quantitative RT-PCR, Concentration Assay, Expressing, Knockdown, Incubation, Negative Control, Western Blot, shRNA, Functional Assay, Blocking Assay, Control, Mass Spectrometry, Immunoprecipitation, Phospho-proteomics, Injection, Two Tailed Test

Journal: Nature Communications

Article Title: Galectin-3-integrin α5β1 phase separation disrupted by advanced glycation end-products impairs diabetic wound healing in rodents

doi: 10.1038/s41467-025-62320-w

Figure Lengend Snippet: A – E STZ-induced diabetic rats were i.c . injected with lgals3 adenovirus or control virus (Veh) after wounding, followed by treatment with integrin β1 functional blocking antibody (Anti-β1) or IgG control (IgG). A Representative images of the wounds (left) and percentage of wound closure (right) ( n = 5). B H&E staining of healed wounds. The distance between the first and second yellow dotted lines represents epidermis thickness (red arrows), and the distance between the second and third yellow dotted lines represents granulation thickness (black arrows). Quantifications of the epidermis and granulation thickness were shown on the lower ( n = 5). Scale bar, 500 µm. C Picrosirius red staining showing COL1 and COL3 in healed wound under polarized light. Quantifications of COL1 area percentage and total COL1 and COL3 area were shown on the right ( n = 5). Scale bar, 50 µm. D Immunohistochemical staining of CD31 that marked microvessels (black arrows) in healed wounds. Quantifications of microvessel count per field were shown on the right ( n = 5). Scale bar, 50 µm. E Immunoblot analysis and quantifications of CD31 in healed wounds ( n = 5). F , G STZ-induced diabetic rats were i.c . injected with shRNA targeting integrin α5 (shα5) or non-targeting shRNA (shscr) 2 weeks before wounding, following i.c . injected with recombinant lgals3 adenovirus (OE-Gal-3) or control virus (Veh). Normal rats (Normal) were set as negative control. F COL1 and COL3 in picrosirius red staining in healed wounds. Quantifications of COL1 area percentage and total COL1 and COL3 area were shown ( n = 5). Scale bar, 50 µm. G Immunohistochemical staining of CD31 (black arrows) in healed wounds. Quantifications of microvessel count per field were shown on the right ( n = 5). Scale bar, 50 µm. H Recombinant Gal-3-induced migration of HMEC-1 cells treated with FAK inhibitor (25 μM), Src-inhibitor (1 μM) or IKK inhibitor (0.5 μM) for 48 h. (Veh, n = 5 biological replicates; FAK inhibitor, n = 3 biological replicates; Src-inhibitor, n = 5 biological replicates; IKK inhibitor, n = 5 biological replicates). I Immunoblot analysis of the phosphorylation of FAK (p-FAK, Y397) in HMEC-1 cells treated with recombinant Gal-3 (10 µg/mL). p-FAK levels were normalized to FAK. Quantifications were shown below the blots. J Recombinant Gal-3-induced tube formation of HMEC-1 cells treated with FAK inhibitor (FAKi, 25 μM), ( n = 4 biological replicates). K Immunoblot analysis (upper) and quantifications (lower) of p-FAK in HMEC-1 cells treated with si-integrin β1 (si-β1) or negative control. p-FAK levels were normalized to FAK ( n = 3 biological replicates). L , M HFD/STZ mice were i.c . injected with recombinant lgals3 adenovirus (OE-Gal-3) or control virus (Veh) after wounding, followed by treatment with FAKi (OE-Gal-3+ FAKi, 15 µM, 100 µL/mouse) or vehicle once every two days. L COL1 and COL3 in picrosirius red staining in healed wounds (left). Quantifications of COL1 area percentage and total COL1 and COL3 area (right). Scale bar, 50 µm. M Immunohistochemical staining and quantifications of CD31 (black arrows) in healed wounds. ( n = 3, two sections per mouse). Scale bar, 50 µm. All statistical data are presented as means ± SEM. P values were determined by unpaired two-tailed Student’s t -test ( A – H , J – M ) or one-way ANOVA with Fisher’s LSD post hoc test ( H ). Error bars mean ± SEM of each group. * P < 0.05; ** P < 0.01; *** P < 0.001. Source data are provided as a Source Data file. Exact p values are provided in the Source Data file.

Article Snippet: Purified GFP-Gal-3 (80 μM) was mixed with integrin β1 (400 nM) (10587-H08H1, Sino Biological, China), integrin α5β1 (400 nM), integrin β1 (400 nM) + lactose (20 mM), integrin α5β1 (400 nM) + lactose (20 mM), or CD146 (400 nM) in PBS and incubated at 37 °C for 5 min. PNGase F-treated integrin α5β1 was prepared by incubating 4 μg of integrin α5β1 with 1000 U of PNGase F at 37 °C for 2 h using a PNGase F Kit (P0704S, NEW ENGLAND BioLabs, China) according to the manufacturer’s instructions.

Techniques: Injection, Control, Virus, Functional Assay, Blocking Assay, Staining, Immunohistochemical staining, Western Blot, shRNA, Recombinant, Negative Control, Migration, Phospho-proteomics, Two Tailed Test

Journal: Nature Communications

Article Title: Galectin-3-integrin α5β1 phase separation disrupted by advanced glycation end-products impairs diabetic wound healing in rodents

doi: 10.1038/s41467-025-62320-w

Figure Lengend Snippet: A Confocal microscopy of integrin α5 segregation in HMEC-1 cells treated with recombinant Gal-3 (1.65 µM) plus lactose (10 mM). An enlarged view of the boxed region shows clusters on the cell membrane (red arrows). Quantifications of cluster number per cell (left to right, n = 5, 6, 5 fields; total number of cells were 50-90 in each group). Scale bar, 20 µm. B , C Condensates formed with the GFP-Gal-3 (80 µM), GFP-Gal-3 (80 µM) + integrin β1 (400 nM) mixture, GFP-Gal-3 (80 µM) + integrin β1 (400 nM) mixture plus lactose (20 mM), and GFP-Gal-3 (80 µM) + CD146 (400 nM) mixture in PBS (pH 7.4), respectively. B Fluorescence microscopy images of the condensates (red arrows) and quantifications of condensates’ number and diameter in each microscope field ( n = 5 biological replicates). Scale bar, 10 µm. C Solution turbidity for the indicated mixtures measurements by UV-vis spectrophotometry ( n = 3 biological replicates each group). D , E Condensates formed with the GFP-Gal-3 (80 µM), GFP-Gal-3 (80 µM) + integrin α5β1 (400 nM) mixture, GFP-Gal-3 (80 µM) + integrin α5β1 (400 nM) mixture plus lactose (20 mM), and GFP-Gal-3 (80 µM) + CD146 (400 nM) mixture in PBS (pH 7.4). D Fluorescence microscopy images of the condensates and quantifications of condensates’ number, and diameter of each microscope field were shown ( n = 5 biological replicates). Scale bar, 10 µm. E Solution turbidity for the mixtures (left to right, n = 3, 4, 3, 3 biological replicates). F Solution turbidity for the mixtures formed with Gal-3 (0 µM, 10 µM, 20 μM, 40 µM, 80 µM and 100 µM) and integrin α5β1 (400 nM) ( n = 3 biological replicates). G Fluorescence Recovery After Photobleaching (FRAP) analysis of droplets formed with GFP-Gal-3 (80 µM) and integrin β1 (400 nM), integrin α5β1 (400 nM) or CD146 (400 nM), respectively. Representative confocal microscopy images (left) and normalized fluorescence intensity (right) after bleaching were shown ( n = 5, 4, 5 independent measurements, respectively). H Condensates formed with the GFP-Gal-3 (80 µM) and integrin α5β1 (400 nM) in PBS (pH 7.4) had their N-glycans removed by PNGase. Fluorescence microscopy images of the condensates and quantifications of condensates’ number, and diameter of each microscope field ( n = 4 biological replicates). Scale bar, 10 µm. I Confocal microscopy of Gal-3 (3.3 µM, containing 0.8 µM GFP-Gal-3 and 2.5 µM Gal-3) induced condensates in CHO-K1 cells expressing the mCherry-integrin α5, with or without 1, 6-hexanediol (1, 6-HD) (1.5%, 2 min). An enlarged view of the boxed region was shown on the right, with cross-sectional fluorescence intensity profiles along the white dotted line in histograms demonstrating the correlation between the two signals. Quantifications of the size and the number of the condensates per cell were shown (upper, n = 5, 5, 5 fields; lower, n = 5, 5, 4 fields, total number of cells were 60-70 in each group). Scale bar, 20 μm. J FRAP measurements. The co-localized Gal-3/integrin α5 condensates in living cells were randomly selected for bleaching (upper panel). Enlarged views of the boxed region were shown. Representative confocal microscopy images (middle panel) and normalized fluorescence intensity (lower panel) after bleaching ( n = 5 independent measurements). Scale bar, 10 μm. K Immunoblot analysis and quantifications of the phosphorylation of FAK (p-FAK, Y397) in HMEC-1 cells treated with Gal-3 (0.33 µM, 15 min) together with PBS, lactose (4 mM), or 1, 6-HD (1.5%, 2 min) in the indicated group ( n = 6, 5, 6, 6 biological replicates). L Confocal microscopy of Gal-3 (3.3 µM, containing 0.8 µM GFP-Gal-3 and 2.5 µM Gal-3) induced condensates in CHO-K1 cells co-expressing mCherry-integrin α5 and mTagBFP2-CD146 treated with lactose (10 mM), sucrose (10 mM) or 1, 6-HD (1.5%, 2 min). Enlarged views of the boxed region were shown with corresponding cross-sectional fluorescence intensity profiles along the white dotted line in histograms demonstrating the correlation between the three signals. Quantifications of the size and number of the condensates per cell were shown ( n = 5 fields, total number of cells was 40–80 in each group). Scale bar, 20 μm. M Recombinant Gal-3-induced tube formation in HMEC-1 cells treated with siRNA targeting integrin β1 or CD146 ( n = 3 biological replicates). All statistical data are presented as means ± SEM. P values were determined by unpaired two-tailed Student’s t test ( A , H , M ), one-way ANOVA with Fisher’s LSD post hoc test ( C , E , F , K ), two-sided Mann-Whitney U test ( I , L ) or Kruskal–Wallis test with Dunn’s post hoc test ( B , D ). Error bars mean ± SEM of each group. * P < 0.05; ** P < 0.01; *** P < 0.001. Source data are provided as a Source Data file. Exact p values are provided in the Source Data file.

Article Snippet: Purified GFP-Gal-3 (80 μM) was mixed with integrin β1 (400 nM) (10587-H08H1, Sino Biological, China), integrin α5β1 (400 nM), integrin β1 (400 nM) + lactose (20 mM), integrin α5β1 (400 nM) + lactose (20 mM), or CD146 (400 nM) in PBS and incubated at 37 °C for 5 min. PNGase F-treated integrin α5β1 was prepared by incubating 4 μg of integrin α5β1 with 1000 U of PNGase F at 37 °C for 2 h using a PNGase F Kit (P0704S, NEW ENGLAND BioLabs, China) according to the manufacturer’s instructions.

Techniques: Confocal Microscopy, Recombinant, Membrane, Fluorescence, Microscopy, Spectrophotometry, Expressing, Western Blot, Phospho-proteomics, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Galectin-3-integrin α5β1 phase separation disrupted by advanced glycation end-products impairs diabetic wound healing in rodents

doi: 10.1038/s41467-025-62320-w

Figure Lengend Snippet: A Tube formation induced by recombinant Gal-3 in HUVECs with diabetic or non-diabetic patient serum (7.5%, v/v) (Non-DM, n = 5; DM, n = 6 biological replicates). B Recombinant Gal-3-induced tube formation in HUVECs treated with different concentrations of BSA-conjugated AGEs (0, 1, 10, 100 µg/mL) was normalized to the group treated with the corresponding concentration of BSA alone ( n = 3 biological replicates). C Immunoblot analysis of phosphorylated-integrin β1 (p-integrin β1) in HMEC-1 cells with Gal-3 (0.33 µM) in the presence or absence of BSA (1.98 µM) or AGEs (1.98 µM). Quantifications were shown below the blots. D Pull-down assays. HMEC-1 cell lysates (100 μg) were pulled down with His-Gal-3 in the presence or absence of BSA or AGEs, the molar ratio of Gal-3 with BSA or AGEs was 1:6. Immunoblot analysis and quantifications of integrin β1 were shown. E Chemical shift changes (Δδ) from these HSQC spectra of Gal-3 and integrin β1. 1 H- 15 N HSQC spectral expansions for 15 N-enriched Gal-3 (20 μM) in the presence of integrin β1 (0.4 μM), plus AGEs (0.4 μM, lower panel). F Bio-Layer interferometry (BLI) analysis of Gal-3-integrin β1 affinity. His-integrin β1 interacted with Gal-3 (200, 400, 600, 800, 1000, 1200, 1400 nM) (left) or Gal-3 (1.4 µM), respectively, plus different concentrations of AGEs (0, 11.2, 22.4, 44.8 µM) (right) at 25 °C. G Representative fluorescence images of condensates formed with GFP-Gal-3 (40 µM) plus BSA or AGEs (240 µM), GFP-Gal-3 (40 µM) + integrin β1 (400 nM) mixture plus BSA or AGEs (240 µM) in PBS (pH 7.4). Quantifications of condensates’ number and diameter in each microscope field were shown ( n = 4 biological replicates). Scale bar, 10 µm. H Particle size of condensates formed by Gal-3 (40 µM) and integrin β1 (400 nM) plus BSA (240 µM) or AGEs (240 µM) in PBS (pH 7.4) ( n = 3 biological replicates). I Confocal microscopy of GFP-Gal-3 (3.3 µM) induced condensates in CHO-K1 cells expressing mCherry-integrin α5 with treatment of BSA (19.8 µM) or AGEs (19.8 µM). The cell indicated by the white arrow was enlarged. Quantifications of the size and the number of the condensates per cell were shown (upper, left to right, n = 5, 6, 6, 5 fields; lower, n = 5 fields; total number of cells were 50–80 in each group). Scale bar, 20 μm. J , K STZ-induced diabetic rats were treated with hydrogels embedded AGEs inhibitor (DM + AGEi) or vehicle (DM + Veh) after wounding. Normal rats treated with blank hydrogels (Normal + Veh) served as the negative control. J Representative images of wounds and percentage of wound closure ( n = 6). K Immunohistochemical staining of CD31(black arrows) in healed wounds. Quantifications of microvessel count per field were shown on the right (left to right, n = 6, 7, 5). Scale bar, 50 µm. All statistical data are presented as means ± SEM. P values were determined by unpaired two-tailed Student’s t test ( A , G , H , J , K ), one-way ANOVA with two-sided Fisher’s LSD post hoc test ( B ) or two-sided Mann–Whitney U test ( I ). Error bars represent the mean ± SEM of each group. * P < 0.05; ** P < 0.01; *** P < 0.001. Source data are provided as a Source Data file. Exact p values are provided in the Source Data file.

Article Snippet: Purified GFP-Gal-3 (80 μM) was mixed with integrin β1 (400 nM) (10587-H08H1, Sino Biological, China), integrin α5β1 (400 nM), integrin β1 (400 nM) + lactose (20 mM), integrin α5β1 (400 nM) + lactose (20 mM), or CD146 (400 nM) in PBS and incubated at 37 °C for 5 min. PNGase F-treated integrin α5β1 was prepared by incubating 4 μg of integrin α5β1 with 1000 U of PNGase F at 37 °C for 2 h using a PNGase F Kit (P0704S, NEW ENGLAND BioLabs, China) according to the manufacturer’s instructions.

Techniques: Recombinant, Concentration Assay, Western Blot, Fluorescence, Microscopy, Confocal Microscopy, Expressing, Negative Control, Immunohistochemical staining, Staining, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Galectin-3-integrin α5β1 phase separation disrupted by advanced glycation end-products impairs diabetic wound healing in rodents

doi: 10.1038/s41467-025-62320-w

Figure Lengend Snippet: In circulation, Gal-3 directly interacts with integrin α5β1 via glycans in vascular endothelial cells, forming a liquid-liquid phase separation, activating downstream FAK, ultimately promoting angiogenesis and skin wound healing. In diabetic states, accumulated AGEs bind to Gal-3, blocking the activation of the integrin α5β1-FAK axis, resulting in reduced angiogenesis and delayed skin wound healing. This figure was created in BioRender. Chen, S. (2025) https://BioRender.com/4tkiilw .

Article Snippet: Purified GFP-Gal-3 (80 μM) was mixed with integrin β1 (400 nM) (10587-H08H1, Sino Biological, China), integrin α5β1 (400 nM), integrin β1 (400 nM) + lactose (20 mM), integrin α5β1 (400 nM) + lactose (20 mM), or CD146 (400 nM) in PBS and incubated at 37 °C for 5 min. PNGase F-treated integrin α5β1 was prepared by incubating 4 μg of integrin α5β1 with 1000 U of PNGase F at 37 °C for 2 h using a PNGase F Kit (P0704S, NEW ENGLAND BioLabs, China) according to the manufacturer’s instructions.

Techniques: Blocking Assay, Activation Assay

Journal: International Journal of Cancer

Article Title: Integrin β6 expression in colorectal cancer cells promotes liver metastasis through enhanced adhesion to endothelial fibronectin

doi: 10.1002/ijc.35504

Figure Lengend Snippet: ITGB6 expression in different CRC cell lines and ITGB6 knockout cells is related to the adhesion of tumour cells to endothelial cells under static and flow conditions. (A) Lysates of lung EC, liver EC, HUVEC, HT‐29 and DLD1 cells were collected and analysed via western blotting with an anti‐fibronectin antibody. GAPDH was used as a loading control. (B) Lysates of CRC cells were collected and subjected to western blot analysis for β6 expression. GAPDH was used as a loading control. (C) CRC cell adhesion (arrows) to HUVEC under static conditions. Scale bar: 200 μm. (D) Quantitative evaluation of CRC cell adhesion to an HUVEC monolayer. Mann–Whitney U test; *** p < 0.001. (E) Lysates of HT‐29 cells with ITGB6 KO and control cells (NT) were collected, and ITGB6 expression was analysed via western blotting. HeLa and BL‐70 cells were used as a negative control for ITGB6 expression. GAPDH was used as a loading control. (F) Cell surface staining for integrin β6 (green; arrows) and DAPI (blue). Scale bar: 50 μm; inset, 10 μm. (G) Apoptosis was induced with Nutlin‐3, and 24 h later, cells were stained using cleaved caspase‐3 (pink) and cleaved PARP (pink) antibodies and DAPI (green). Scale bar: 125 μm. (H) The cleaved caspase‐3 and cleaved PARP signals relative to the DAPI signals observed in G are reported as percentages. Mann–Whitney U test; *** p < 0.001. (I) A scratch was made in a confluent layer of HT‐29 cells. Cells were stimulated with hepatocyte growth factor (HGF), and the wound was allowed to close for 24 h. Scale bar: 500 μm. (J) Quantitative evaluation of the results shown in I. Unpaired t ‐test; ** p < 0.01. (K) Lysates of HT‐29 cells stimulated for 24 or 72 h with 50 ng/μL TNF‐α were collected and analysed via western blotting for β6 expression. The results of the quantification of the integrin β6 protein signals relative to those of GAPDH are given in the numbers below. (L) The capacity of TNF‐α‐treated HT‐29 KO and NT cells to adhere to an HUVEC monolayer after 2 h of incubation. Integrity of the HUVEC monolayer was controlled by phase contrast microscopy (insets). Scale bar: 250 μm. Mann–Whitney U test; ns, not significant; *** p < 0.001; **** p < 0.0001. (M) Representation and quantification of the capacity of ITGB6 KO and NT cells to adhere to an HUVEC monolayer under flow conditions. Arrows indicate the adherent cells. Scale bar: 250 μm. Mann–Whitney U test; *** p < 0.001. In C, D, L, and M, IL‐1β‐stimulated HUVECs were used. In C, L, and M, CRC cells were stained using Cytopainter.

Article Snippet: The following antibodies were used for detection: rabbit anti‐human integrin αv (ab179475, Abcam, RRID: AB_2716738, 1:2000), rabbit anti‐human integrin β1 (12594‐1‐AP, Proteintech, Rosemont, IL, USA, RRID: AB_2130085, 1:5000), rabbit anti‐human integrin β5 (4708S, Cell Signaling, Danvers, MA, USA, RRID: AB_2129156, 1:1000), sheep anti‐human integrin β6 (AF4155, R&D Systems, RRID: AB_2129295, 1:2000), and rabbit anti‐human fibronectin (F3648, Merck, RRID: AB_476976, 1:1000).

Techniques: Expressing, Knock-Out, Western Blot, Control, MANN-WHITNEY, Negative Control, Staining, Incubation, Microscopy

Journal: International Journal of Cancer

Article Title: Integrin β6 expression in colorectal cancer cells promotes liver metastasis through enhanced adhesion to endothelial fibronectin

doi: 10.1002/ijc.35504

Figure Lengend Snippet: Endothelial fibronectin and integrin β6 from tumour cells colocalize during adhesion. (A) HUVEC were fixed and subjected to immunofluorescent staining for fibronectin (red, arrows) and Draq5 (purple). Scale bar: 50 μm. (B) Tumour cells adherent to HUVEC were fixed and subjected to immunofluorescent staining for fibronectin (red), integrin β6 (green, white arrows) and Draq5 (purple). Colocalization of integrin β6 and fibronectin is indicated by yellow arrows. Scale bar: 10 μm. (C) Orthogonal sectioning of the HUVEC and adherent tumour cells confirms the colocalization (yellow arrows) of fibronectin (red) and integrin β6 (green, white arrows). Scale bar: 10 μm; enlarged image: 2 μm.

Article Snippet: The following antibodies were used for detection: rabbit anti‐human integrin αv (ab179475, Abcam, RRID: AB_2716738, 1:2000), rabbit anti‐human integrin β1 (12594‐1‐AP, Proteintech, Rosemont, IL, USA, RRID: AB_2130085, 1:5000), rabbit anti‐human integrin β5 (4708S, Cell Signaling, Danvers, MA, USA, RRID: AB_2129156, 1:1000), sheep anti‐human integrin β6 (AF4155, R&D Systems, RRID: AB_2129295, 1:2000), and rabbit anti‐human fibronectin (F3648, Merck, RRID: AB_476976, 1:1000).

Techniques: Staining

Journal: International Journal of Cancer

Article Title: Integrin β6 expression in colorectal cancer cells promotes liver metastasis through enhanced adhesion to endothelial fibronectin

doi: 10.1002/ijc.35504

Figure Lengend Snippet: Integrin β6 is a proteolysis‐resistant mediator of CRC tumour cell adhesion to endothelial cells. (A) Lysates of HT‐29 and DLD‐1 cells harvested in the presence of 0.05% trypsin or 5 mM EDTA‐PBS were collected, and the expression of integrins αv, β6, β1 and β5 was analysed using western blotting. Blots show the full‐length proteins as well as the trypsin‐induced protein fragments. GAPDH was used as a loading control. (B) Adhesion of cytopainter‐stained ITGB6 KO or NT cells harvested by trypsinization or 5 mM PBS‐EDTA to an IL‐1β‐stimulated HUVEC monolayer under static conditions. Arrow points towards an adherent cell. Scale bar: 250 μm. The quantification is shown below. Mann–Whitney U test; ns, not significant; ** p < 0.01. (C) The cancer‐related survival (60‐month cut‐off) of CRC patients who did not receive neoadjuvant therapy and had no residual tumour (R0) was explored. Based on MMP11 and MMP12 RNA expression levels, patients were divided into high‐ and low‐MMP groups. ITGB6 ‐dependent survival was investigated in these groups. The number of patients per group is indicated by ‘ n ’. Cox proportional hazards survival regression. (D) Recombinant integrins αvβ6, αvβ1, and αvβ5 were incubated with MMP‐12 at an enzyme‐to‐substrate ratio of 1:10 for 1 h. Proteolytic cleavage of integrins was evaluated via western blotting.

Article Snippet: The following antibodies were used for detection: rabbit anti‐human integrin αv (ab179475, Abcam, RRID: AB_2716738, 1:2000), rabbit anti‐human integrin β1 (12594‐1‐AP, Proteintech, Rosemont, IL, USA, RRID: AB_2130085, 1:5000), rabbit anti‐human integrin β5 (4708S, Cell Signaling, Danvers, MA, USA, RRID: AB_2129156, 1:1000), sheep anti‐human integrin β6 (AF4155, R&D Systems, RRID: AB_2129295, 1:2000), and rabbit anti‐human fibronectin (F3648, Merck, RRID: AB_476976, 1:1000).

Techniques: Expressing, Western Blot, Control, Staining, MANN-WHITNEY, RNA Expression, Recombinant, Incubation

Journal: International Journal of Cancer

Article Title: Integrin β6 expression in colorectal cancer cells promotes liver metastasis through enhanced adhesion to endothelial fibronectin

doi: 10.1002/ijc.35504

Figure Lengend Snippet: Integrin β6 is required for HT‐29 cell adhesion to fibronectin. (A) HT‐29 cells were plated on surfaces coated with the indicated fibronectin concentrations (μg/ml) for 4 h. Scale bar: 250 μm. Mann–Whitney U test; *** p < 0.001. (B) HT‐29 cells were plated on surfaces coated with 30 μg/mL fibronectin, 30 μg/mL vitronectin or 30 μg/mL tenascin C plus 0.1 μg/mL fibronectin for 4 h. Scale bar: 250 μm. Mann–Whitney U test; **** p < 0.0001. (C) HT‐29 cells were pretreated for 10 min with an isotype control (anti‐mouse IgG2a), anti‐αvβ6 antibody or RGD peptide before being plated on surfaces coated with 30 μg/mL fibronectin for 4 h in the presence of the treatment. Scale bar: 250 μm. Mann–Whitney U test; ns, not significant; *** p < 0.001. CRC cells were stained using Cytopainterr. Adherent cells are marked by arrows.

Article Snippet: The following antibodies were used for detection: rabbit anti‐human integrin αv (ab179475, Abcam, RRID: AB_2716738, 1:2000), rabbit anti‐human integrin β1 (12594‐1‐AP, Proteintech, Rosemont, IL, USA, RRID: AB_2130085, 1:5000), rabbit anti‐human integrin β5 (4708S, Cell Signaling, Danvers, MA, USA, RRID: AB_2129156, 1:1000), sheep anti‐human integrin β6 (AF4155, R&D Systems, RRID: AB_2129295, 1:2000), and rabbit anti‐human fibronectin (F3648, Merck, RRID: AB_476976, 1:1000).

Techniques: MANN-WHITNEY, Control, Staining

Journal: International Journal of Cancer

Article Title: Integrin β6 expression in colorectal cancer cells promotes liver metastasis through enhanced adhesion to endothelial fibronectin

doi: 10.1002/ijc.35504

Figure Lengend Snippet: Blocking integrin β6 reduces cancer cell adhesion to endothelial cells. (A) Adhesion of HT‐29 cells to an HUVEC monolayer in the presence of the RGD peptide at the indicated concentrations after 2 h of incubation. Scale bar: 250 μm. Mann–Whitney U test; ns, not significant; *** p < 0.001. (B) Adhesion of pretreated HT‐29 cells to a HUVEC monolayer in the presence of 10 μg/mL anti‐αvβ6 antibody after 2 h of incubation. Scale bar: 250 μm. Mann–Whitney U test; *** p < 0.001. (C) Adhesion of ITGB6 ‐expressing CRC cells (SW948, HT‐29) on an HUVEC monolayer can be blocked by 10 μg/mL RGD peptide and 10 μg/mL anti‐αvβ6 antibody. CRC cell lines with low ITGB6 expression (LOVO, RKO) exhibit low adhesion to HUVEC. Scale bar: 250 μm. Mann–Whitney U test; ns, not significant; *** p < 0.001. In A, B, and C, IL‐1β‐stimulated HUVEC were used, and CRC cells were stained using cytopainter. Adherent cells are indicated by arrows.

Article Snippet: The following antibodies were used for detection: rabbit anti‐human integrin αv (ab179475, Abcam, RRID: AB_2716738, 1:2000), rabbit anti‐human integrin β1 (12594‐1‐AP, Proteintech, Rosemont, IL, USA, RRID: AB_2130085, 1:5000), rabbit anti‐human integrin β5 (4708S, Cell Signaling, Danvers, MA, USA, RRID: AB_2129156, 1:1000), sheep anti‐human integrin β6 (AF4155, R&D Systems, RRID: AB_2129295, 1:2000), and rabbit anti‐human fibronectin (F3648, Merck, RRID: AB_476976, 1:1000).

Techniques: Blocking Assay, Incubation, MANN-WHITNEY, Expressing, Staining

Journal: International Journal of Cancer

Article Title: Integrin β6 expression in colorectal cancer cells promotes liver metastasis through enhanced adhesion to endothelial fibronectin

doi: 10.1002/ijc.35504

Figure Lengend Snippet: ITGB6 expression is correlated with tumour stage and highly increased in liver metastases of colorectal cancer. (A) The association of ITGB6 expression with the UICC 2017 stages of 464 CRC patients. Mann–Whitney U test; * p < 0.05; ** p < 0.01. (B) Different grades of IHC staining for integrin β6 in primary CRC tissues. Staining signals are indicated by an arrow. Scale bar: 500 μm. (C) Comparison of ITGB6 expression measured in CRC tissues ( n = 14) using qRT–PCR and IHC. (D) IHC staining for integrin β6 in primary CRC tissue and liver metastatic lesions from the same patients ( n = 19). Scale bar: 250 μm; enlarged image: 50 μm. (E) Comparison of integrin β6 expression in primary CRC tissues and liver metastases from the same patients. 0, negative; 1, low expression; 2, medium expression; 3, high expression. Increased expression in the liver metastatic tissue compared with the CRC tissue is indicated in blue. No change in expression is noted in black. Decreased expression is noted in red.

Article Snippet: The following antibodies were used for detection: rabbit anti‐human integrin αv (ab179475, Abcam, RRID: AB_2716738, 1:2000), rabbit anti‐human integrin β1 (12594‐1‐AP, Proteintech, Rosemont, IL, USA, RRID: AB_2130085, 1:5000), rabbit anti‐human integrin β5 (4708S, Cell Signaling, Danvers, MA, USA, RRID: AB_2129156, 1:1000), sheep anti‐human integrin β6 (AF4155, R&D Systems, RRID: AB_2129295, 1:2000), and rabbit anti‐human fibronectin (F3648, Merck, RRID: AB_476976, 1:1000).

Techniques: Expressing, MANN-WHITNEY, Immunohistochemistry, Staining, Comparison, Quantitative RT-PCR