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: After blocking with 5% non-fat dry milk in PBST (PBS containing 0.05% Tween-20), blots were incubated overnight at 4°C with the following primary antibodies: CD31 (1:1000, ab281583, Abcam, USA), β-Actin (1:1000, 612657, BD Biosciences, USA), Gal-3 (1:1000, ab76245, Abcam, USA), collagen I (1:1000, GB114197 , Servicebio, China), collagen III (1:1000, EPR17673 , Abcam, USA), AKT (1:1000, 9272 s, cell signaling technology, USA), p-AKT (Ser473) (1:1000, 9271 s, cell signaling technology, USA), FoxO1 (1:1000, 18592-1-AP, Proteintech, China), integrin β1 (1:1000, 610467, BD Biosciences, USA), integrin α3 (1:1000, 66070, Proteintech, China), integrin α5 (1:1000, sc-10729, Santa Cruz, USA), integrin α6 (1:1000, ab181551, Abcam, USA), p-integrin β1 (Y783) (1:1000, ab62337, Abcam, USA), VEGFR2 (1:1000, 2479S, Cell signaling, USA), p-VEGFR2 (Tyr1175) (1:1000, 2478S, Cell signaling technology, USA), EGFR (1:1000, 4267S, Cell signaling, USA), p-EGFR (Tyr1068) (1:1000, 3777S, Cell signaling technology, USA), integrin αv(1:1000, 27096-1-AP, Proteintech, China), FAK (1:1000, 12636-1-AP, Proteintech China), p-FAK (Y397) (1:1000, 611806, BD Biosciences, USA), CD146 (1:1000, 611208, BD Biosciences, USA), GST (1:1000, KM8005, Sungene Biotech, China), His-tag (1:1000, RM1001, Beijing Ray Antibody Biotech, China), α-Tubulin (1:1000, F0063, Selleck, USA).
Techniques: Confocal Microscopy, Recombinant, Membrane, Fluorescence, Microscopy, Spectrophotometry, Expressing, Western Blot, Phospho-proteomics, Two Tailed Test, MANN-WHITNEY
Santa Cruz Biotechnology is a verified supplier 
![( A ) ITGA3 mRNA expression in normal bladder tissues ( n = 18) and primary bladder tumors ( n = 408) based on TCGA data. ( B ) ITGA3 expression across consensus molecular subtypes of bladder cancer from five aggregated clinical cohorts. ( C ) Representative multiplex immunofluorescence images of TMA cores stained for ITGA3 (green), ITGB1 (red), and nuclei [DAPI (4′,6-diamidino-2-phenylindole), blue] of primary bladder carcinomas and normal tissues. Scale bars, 100 μm. ( D to F ) Quantification of ITGA3 expression in tumor versus normal tissues: fluorescence intensity (D), percentage of ITGA3 -positive cells (E), and H -scores (F). **** P < 0.0001.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_7053/pmc12757053/pmc12757053__sciadv.ady0041-f6.jpg)
