Journal: BMC Biology

Article Title: The phosphatase PPM1F, a negative regulator of integrin activity, is essential for embryonic development and controls tumor cell invasion

doi: 10.1186/s12915-025-02254-3

Figure Lengend Snippet: Increased integrin β1 activity, elevated cell adhesion, and migration defects of ppm1f-/- MEFs are reverted by re-expression of wildtype PPM1F. A PPM1F-/- MEFs were transduced with lentiviral particles encoding human wildtype PPM1F (hWT) or human PPM1F D360 A (hDA) in a bi-cistronic expression cassette with GFP. In addition, PPM1F-/- MEFs and PPM1F +/+ cells were transduced with a lentivirus encoding GFP alone. WCLs of sorted cells were analyzed by Western blotting with the indicated antibodies; as controls, WCLs of 293 T cells transfected with the empty vector (mock), GFP (GFP) or murine PPM1F (mWT) were loaded. B MEFs as in ( A ) were seeded onto 1 µg/ml FN III9-12 for 2 h. Samples were fixed and stained for talin (upper panel) or the active integrin β1 (lower panel) before analysis by confocal microscopy; scale bar: 20 µm. Insets show higher magnification of boxed areas; scale bar: 5 µm. Arrowheads point to active integrin β1 or talin enrichment. C MEFs as in ( A ) were kept in suspension for 45 min and incubated for 15 min with 10 µg/ml FN III9-12 (FN). Samples were stained for total (Hmb1-1) or active β1 integrin (9EG7) and analyzed by flow cytometry, ≥ 10 000 counts. The mean fluorescence intensity (MFI) ratio of active to total β1 integrin was calculated and normalized to the wildtype sample (= 1). Scatter blots represent mean ± SEM of 4 independent experiments; statistics was performed using one-way ANOVA and Bonferroni post-hoc test ( p *** < 0.001, ns = not significant). D MEFs were seeded in triplicates onto fibronectin-coated wells for 60 min and cell adhesion was quantified. Representative pictures from cells seeded on 10 µg/ml FN (left panel); scale bar: 150 µm. Scatter blots represent mean ± SEM of 5 independent experiments performed in technical triplicates each. Values were normalized to MEF wildtype cells (= 1). Statistics was performed using one-way ANOVA, followed by Bonferroni post-hoc test (** p < 0.01, * p < 0.05, ns = not significant). E MEFs were seeded onto indicated fibronectin concentrations for 45 min, fixed and stained with DAPI and Phalloidin-Cy5. Samples were imaged using confocal microscopy. Representative images from cells seeded onto 10 µg/ml FN are shown; scale bar: 10 µm (left panel). Quantification of cell spreading. Boxes and whiskers indicate median with 95% confidence intervals from 2 independent experiments; n ≥ 90 cells. Statistics was performed using one-way ANOVA, followed by Bonferroni post-hoc test (*** p < 0.001, ns = not significant) (right panel). F Serum starved MEFs were stimulated by addition of 10% FCS and cell migration was monitored every 30 min for 12 h using time-lapse microscopy. Cell tracks were evaluated for velocity, covered distance and directionality. Boxes and whiskers indicate median with 95% confidence intervals from 2 independent experiments ( n = 30); Statistics was performed as in ( E ); *** p < 0.001, * p < 0.05, ns = not significant. See also Additional_File2

Article Snippet: The following antibodies were used with the corresponding dilutions for western blot analysis (WB), immunofluorescence (IF), immunohistochemistry (IHC), immunoprecipitation (IP), or integrin activity assay (IA): α-Actinin (BM75.2, mouse anti-human, Abcam; 1:1000 WB), α 1 -integrin (TS2/7, mouse anti-human/anti-mouse, Abcam; 1:50 IF), α 2 -integrin (6 F1, mouse anti-human/anti-mouse, DSHB; 1:60 IF), α 3 -integrin (P1B5, mouse anti-human/anti-mouse, DSHB; 1:60 IF), α 5 -integrin (BIIG2, rat anti-human/anti-mouse, DSHB; 1:10 IF), α v -integrin (PE-P2 W7 mouse anti-human/anti-mouse, sc-9969; IF 1:300), β 1 -integrin (HMβ1-1, armenian hamster anti-mouse, Bio Legend; 1:300 IF; AIIB2, rat anti-human/anti-mouse, DSHB; 1:600 IF, IA; M-106, rabbit anti-mouse/anti-human, Santa Cruz; 1:500 WB; D2E5, rabbit anti-human, Cell Signaling; 1:1000 WB), human β 1 -integrin (P5D2, mouse anti-human, DSHB, 2.5 μg IP; 9EG7, rat anti- human, DSHB 2.5 μg IP; AIIB2, rat anti-human, DSHB; 2.5 μg IP), β 3 -integrin (2 C9.G3, arm. hamster anti-mouse, eBioscience; 1:300 IF; PM6/13, mouse anti-human, Abcam; 1:100 IF), β 5 -integrin (KN-52, mouse anti-mouse/human, eBioscience; IF 1:300), Focal adhesion kinase (FAK) (77, mouse anti-human, BD; 1:250 WB), integrin-linked kinase (ILK) (EP1593Y, rabbit anti-human, Epitomics; 1:800 WB), Kindlin-2 (3 A3, mouse anti-human, Millipore; 1:200 WB, 1:250 IF), Laminin (ab11575, rabbit anti-mouse, Abcam; 1:300 IHC), Nestin (rat-401, anti-mouse, Millipore; IHC 1:200), Paxillin (5H11, mouse monoclonal, Thermo Scientific; 1:1000 WB), hPPM1F (17,020–1-AP, rabbit anti-human, Protein-Tech; 1:1000 WB), mPPM1F (#1147, rabbit anti-mouse PPM1F; generated at the central animal care facility; University of Konstanz; 1:200 WB; see Additional File2: Fig. S2), FilaminA (EP2405Y, IgG, rabbit anti-human, Epitomics; 1:125.000 WB), Tubulin (E7, IgG1, mouse anti-human, DSHB; 1:1000), Talin (8 d4, mouse anti-human, Thermo Scientific; 1:800 WB, 1:40 IF), Vinculin (hVIN-1, mouse anti-human, Sigma; 1:2000 WB, 1:200 IF), Zyxin (Zol301, mouse anti-human, Abcam; 1:1000 WB), Dylight488-conjugated goat anti-mouse IgG (Jackson; 1:200), Cy3-conjugated goat anti-rabbit IgG (Jackson; 1:200), Cy3-conjugated goat anti-mouse IgG (Jackson; 1:200), Cy5-conjugated goat anti-mouse IgG (Jackson; 1:200), RhodamineRed-conjugated goat anti-rat IgG (Jackson; 1:200), RhodamineRed-conjugated goat anti-Armenian Hamster IgG (Jackson; 1:200), HRP-conjugated goat anti-mouse IgG (Jackson; WB 1:10 000), HRP-conjugated goat anti-rat IgG (Santa Cruz; 1:250), HRP-conjugated goat anti-rabbit IgG (Jackson; WB 1:3000), unspecific control IgG (anti-mouse, 96/1, generated at the Tierforschungsanlage; University of Konstanz; anti-rat, MJ7/18 Endoglin, DSHB).

Techniques: Activity Assay, Migration, Expressing, Transduction, Western Blot, Transfection, Plasmid Preparation, Staining, Confocal Microscopy, Suspension, Incubation, Flow Cytometry, Fluorescence, Time-lapse Microscopy

Journal: BMC Biology

Article Title: The phosphatase PPM1F, a negative regulator of integrin activity, is essential for embryonic development and controls tumor cell invasion

doi: 10.1186/s12915-025-02254-3

Figure Lengend Snippet: PPM1F contributes to the invasive phenotype of tumor cells. A WCLs from indicated cancer cell lines were analyzed by Western blotting with α-human PPM1F or α-integrin β1 antibodies. α-Tubulin antibody was used as loading control. B , C Indicated serum-starved cancer cells were seeded on top of a Matrigel basement membrane (30 µg/100 µl) in Boyden chamber cell invasion assays using 20% FCS as stimulus or 2% BSA to evaluate random invasion activity. NIH3 T3 cells served as non-invasive control cells. Representative pictures of the lower porous membrane surface (20x) are shown in (B); scale bar: 50 µm. Crystal violet-stained cells can be distinguished from the 8 µm membrane pores. Cells were evaluated for invasion after 24 h by dye elution with 10% acetic acid and absorbance measurement at 590 nm. Graph in ( C ) shows quantified means ± SEM from three independent experiments. Statistics was performed using one-way ANOVA and Bonferroni post-hoc test ( p *** < 0.001, p ** < 0.01, ns = not significant). D MCF-7 cells were stably transduced with lentiviral particles harboring a bicistronic GFP and hPPM1F wildtype or hPPM1F D360 A expression cassette and single-cell sorted via flow cytometry for GFP positive cells to obtain a mixed population of PPM1F-overexpressing MCF-7 cells (PPM1F + + and PPM1F D360 A + +). WCL of the wildtype and modified cell lines were analyzed by Western blotting with indicated antibodies. α-tubulin antibody (lowest panel) served as loading control. E Serum-starved cells from ( D ) were seeded on top of a Matrigel base (30 µg/100 µl) in Boyden chambers. Cell invasion was stimulated by addition of 20% FCS or 2% BSA to the lower chamber. Representative pictures of the lower porous membrane surface (20x) are shown; scale bar: 50 µm. Crystal violet-stained cells can be distinguished from the 8 µm membrane pores. Invasion was quantified by dye elution. Graph (right) shows means ± SEM from four independent experiments performed in triplicate. Statistics as in ( C )

Article Snippet: The following antibodies were used with the corresponding dilutions for western blot analysis (WB), immunofluorescence (IF), immunohistochemistry (IHC), immunoprecipitation (IP), or integrin activity assay (IA): α-Actinin (BM75.2, mouse anti-human, Abcam; 1:1000 WB), α 1 -integrin (TS2/7, mouse anti-human/anti-mouse, Abcam; 1:50 IF), α 2 -integrin (6 F1, mouse anti-human/anti-mouse, DSHB; 1:60 IF), α 3 -integrin (P1B5, mouse anti-human/anti-mouse, DSHB; 1:60 IF), α 5 -integrin (BIIG2, rat anti-human/anti-mouse, DSHB; 1:10 IF), α v -integrin (PE-P2 W7 mouse anti-human/anti-mouse, sc-9969; IF 1:300), β 1 -integrin (HMβ1-1, armenian hamster anti-mouse, Bio Legend; 1:300 IF; AIIB2, rat anti-human/anti-mouse, DSHB; 1:600 IF, IA; M-106, rabbit anti-mouse/anti-human, Santa Cruz; 1:500 WB; D2E5, rabbit anti-human, Cell Signaling; 1:1000 WB), human β 1 -integrin (P5D2, mouse anti-human, DSHB, 2.5 μg IP; 9EG7, rat anti- human, DSHB 2.5 μg IP; AIIB2, rat anti-human, DSHB; 2.5 μg IP), β 3 -integrin (2 C9.G3, arm. hamster anti-mouse, eBioscience; 1:300 IF; PM6/13, mouse anti-human, Abcam; 1:100 IF), β 5 -integrin (KN-52, mouse anti-mouse/human, eBioscience; IF 1:300), Focal adhesion kinase (FAK) (77, mouse anti-human, BD; 1:250 WB), integrin-linked kinase (ILK) (EP1593Y, rabbit anti-human, Epitomics; 1:800 WB), Kindlin-2 (3 A3, mouse anti-human, Millipore; 1:200 WB, 1:250 IF), Laminin (ab11575, rabbit anti-mouse, Abcam; 1:300 IHC), Nestin (rat-401, anti-mouse, Millipore; IHC 1:200), Paxillin (5H11, mouse monoclonal, Thermo Scientific; 1:1000 WB), hPPM1F (17,020–1-AP, rabbit anti-human, Protein-Tech; 1:1000 WB), mPPM1F (#1147, rabbit anti-mouse PPM1F; generated at the central animal care facility; University of Konstanz; 1:200 WB; see Additional File2: Fig. S2), FilaminA (EP2405Y, IgG, rabbit anti-human, Epitomics; 1:125.000 WB), Tubulin (E7, IgG1, mouse anti-human, DSHB; 1:1000), Talin (8 d4, mouse anti-human, Thermo Scientific; 1:800 WB, 1:40 IF), Vinculin (hVIN-1, mouse anti-human, Sigma; 1:2000 WB, 1:200 IF), Zyxin (Zol301, mouse anti-human, Abcam; 1:1000 WB), Dylight488-conjugated goat anti-mouse IgG (Jackson; 1:200), Cy3-conjugated goat anti-rabbit IgG (Jackson; 1:200), Cy3-conjugated goat anti-mouse IgG (Jackson; 1:200), Cy5-conjugated goat anti-mouse IgG (Jackson; 1:200), RhodamineRed-conjugated goat anti-rat IgG (Jackson; 1:200), RhodamineRed-conjugated goat anti-Armenian Hamster IgG (Jackson; 1:200), HRP-conjugated goat anti-mouse IgG (Jackson; WB 1:10 000), HRP-conjugated goat anti-rat IgG (Santa Cruz; 1:250), HRP-conjugated goat anti-rabbit IgG (Jackson; WB 1:3000), unspecific control IgG (anti-mouse, 96/1, generated at the Tierforschungsanlage; University of Konstanz; anti-rat, MJ7/18 Endoglin, DSHB).

Techniques: Western Blot, Control, Membrane, Activity Assay, Staining, Stable Transfection, Transduction, Expressing, Flow Cytometry, Modification

Journal: BMC Biology

Article Title: The phosphatase PPM1F, a negative regulator of integrin activity, is essential for embryonic development and controls tumor cell invasion

doi: 10.1186/s12915-025-02254-3

Figure Lengend Snippet: Genetic deletion of PPM1F in tumor cells diminishes matrix invasion and integrin phosphorylation. A WCLs from A172 wildtype cells and two clonal PPM1F KO cell lines (1 and 2) were analyzed by Western blotting using the indicated antibodies. α-Tubulin antibody was used as loading control. B Serum starved A172 wildtype cells and PPM1F KO cell lines (clone 1 and clone 2) were seeded in triplicate onto fibronectin-, vitronectin-, or 2% BSA-coated wells for 60 min either in presence of 50 µM cilengitide or DMSO as control. Wells were washed and adherent cells were stained with crystal violet. Representative pictures are shown; scale bar: 150 µm. C Adherent crystal violett stained cells from ( B ) were quantified by dye elution. Graph depicts individual values as well as mean ± SEM of 4 independent experiments performed in technical triplicates. Statistics was performed using one-way ANOVA, followed by Bonferroni post-hoc test (*** p < 0.001; ** p < 0.01; p * < 0.05; ns = not significant) and shown for the PPM1F knock-out clones in relation to the A172 wildtype cells. D Serum-starved cells as in ( C ) were seeded on top of a Matrigel base (30 µg/100 µl) in Boyden chambers and cell invasion was stimulated by addition of 20% FCS or 2% BSA to the lower chamber. Cells were evaluated for invasion after 24 h and representative pictures of the lower porous membrane surface (20x) are shown; scale bar: 50 µm. Crystal violet-stained cells can be distinguished from the 8 µm membrane pores (left). Invasion assays were quantified by dye elution. Graph depicts individual values as well as means ± SEM from four independent experiments performed in triplicate. Statistics as in ( C ). See also Additional_File4 and Additional_File5

Article Snippet: The following antibodies were used with the corresponding dilutions for western blot analysis (WB), immunofluorescence (IF), immunohistochemistry (IHC), immunoprecipitation (IP), or integrin activity assay (IA): α-Actinin (BM75.2, mouse anti-human, Abcam; 1:1000 WB), α 1 -integrin (TS2/7, mouse anti-human/anti-mouse, Abcam; 1:50 IF), α 2 -integrin (6 F1, mouse anti-human/anti-mouse, DSHB; 1:60 IF), α 3 -integrin (P1B5, mouse anti-human/anti-mouse, DSHB; 1:60 IF), α 5 -integrin (BIIG2, rat anti-human/anti-mouse, DSHB; 1:10 IF), α v -integrin (PE-P2 W7 mouse anti-human/anti-mouse, sc-9969; IF 1:300), β 1 -integrin (HMβ1-1, armenian hamster anti-mouse, Bio Legend; 1:300 IF; AIIB2, rat anti-human/anti-mouse, DSHB; 1:600 IF, IA; M-106, rabbit anti-mouse/anti-human, Santa Cruz; 1:500 WB; D2E5, rabbit anti-human, Cell Signaling; 1:1000 WB), human β 1 -integrin (P5D2, mouse anti-human, DSHB, 2.5 μg IP; 9EG7, rat anti- human, DSHB 2.5 μg IP; AIIB2, rat anti-human, DSHB; 2.5 μg IP), β 3 -integrin (2 C9.G3, arm. hamster anti-mouse, eBioscience; 1:300 IF; PM6/13, mouse anti-human, Abcam; 1:100 IF), β 5 -integrin (KN-52, mouse anti-mouse/human, eBioscience; IF 1:300), Focal adhesion kinase (FAK) (77, mouse anti-human, BD; 1:250 WB), integrin-linked kinase (ILK) (EP1593Y, rabbit anti-human, Epitomics; 1:800 WB), Kindlin-2 (3 A3, mouse anti-human, Millipore; 1:200 WB, 1:250 IF), Laminin (ab11575, rabbit anti-mouse, Abcam; 1:300 IHC), Nestin (rat-401, anti-mouse, Millipore; IHC 1:200), Paxillin (5H11, mouse monoclonal, Thermo Scientific; 1:1000 WB), hPPM1F (17,020–1-AP, rabbit anti-human, Protein-Tech; 1:1000 WB), mPPM1F (#1147, rabbit anti-mouse PPM1F; generated at the central animal care facility; University of Konstanz; 1:200 WB; see Additional File2: Fig. S2), FilaminA (EP2405Y, IgG, rabbit anti-human, Epitomics; 1:125.000 WB), Tubulin (E7, IgG1, mouse anti-human, DSHB; 1:1000), Talin (8 d4, mouse anti-human, Thermo Scientific; 1:800 WB, 1:40 IF), Vinculin (hVIN-1, mouse anti-human, Sigma; 1:2000 WB, 1:200 IF), Zyxin (Zol301, mouse anti-human, Abcam; 1:1000 WB), Dylight488-conjugated goat anti-mouse IgG (Jackson; 1:200), Cy3-conjugated goat anti-rabbit IgG (Jackson; 1:200), Cy3-conjugated goat anti-mouse IgG (Jackson; 1:200), Cy5-conjugated goat anti-mouse IgG (Jackson; 1:200), RhodamineRed-conjugated goat anti-rat IgG (Jackson; 1:200), RhodamineRed-conjugated goat anti-Armenian Hamster IgG (Jackson; 1:200), HRP-conjugated goat anti-mouse IgG (Jackson; WB 1:10 000), HRP-conjugated goat anti-rat IgG (Santa Cruz; 1:250), HRP-conjugated goat anti-rabbit IgG (Jackson; WB 1:3000), unspecific control IgG (anti-mouse, 96/1, generated at the Tierforschungsanlage; University of Konstanz; anti-rat, MJ7/18 Endoglin, DSHB).

Techniques: Phospho-proteomics, Western Blot, Control, Staining, Knock-Out, Clone Assay, Membrane

Journal: BMC Biology

Article Title: The phosphatase PPM1F, a negative regulator of integrin activity, is essential for embryonic development and controls tumor cell invasion

doi: 10.1186/s12915-025-02254-3

Figure Lengend Snippet: Increased integrin-based cell adhesion in PPM1F-deficient cells prohibits cell spreading despite elevated PAK activity. A Serum-starved A172 wildtype, sgRNA control and PPM1F KO cells were seeded onto 2 µg/ml FN III9-12 for 45 min and WCLs were subjected to Western blotting with indicated antibodies (left panel). Graphs (right panel) show densitometric quantification of band intensities from pThr402PAK2 versus PAK antibody signal for the indicated samples from 5 independent experiments; wildtype was set to 1. Statistics were performed using one-way ANOVA, followed by Bonferroni post-hoc test (* p < 0.05, ns = not significant). B Serum-starved A172 wildtype and PPM1F KO cells were seeded onto 2 µg/ml FN III9-12 for 1.5 h, fixed and F-actin was stained. Samples were imaged using confocal microscopy. Representative pictures are shown; scale bar: 20 µm. C Cells as in ( B ) were seeded for 2 h on surfaces coated with 10 µg/ml fibronectin or poly-L-lysine, before fixation, F-actin staining and analysis by confocal microscopy; scale bar: 10 µm. D Spreading assays were performed with serum-starved A172 wildtype and PPM1F KO cells re-expressing mKate2 or re-expressing PPM1F-mKate2 cells, pre-treated with 5 µM DMSO or FRAX597 (PAK1-3 inhibitor) for 45 min in suspension before seeding onto 2 µg/ml FN III9-12 for 1.5 h. Cells were fixed, stained for F-actin and the covered area was quantified in ImageJ. Boxes and whiskers indicate median with 95% confidence intervals from two independent experiments; n ≥ 30 cells; dots indicate outliers. Statistics was performed using one-way ANOVA, followed by post-hoc Bonferroni test, (*** p < 0.001, ns = not significant). E Serum-starved cells as in ( D ) were pre-treated with 5 µM DMSO or FRAX597 (PAK1-3 inhibitor) for 45 min in suspension before seeded onto 2 µg/ml FN III9-12 for 1.5 h. Cells were fixed and stained for active integrin β1. Cells were imaged by confocal microscopy. Representative pictures are shown; scale bar: 10 µm. See also Additional_File6 and Additional_File7

Article Snippet: The following antibodies were used with the corresponding dilutions for western blot analysis (WB), immunofluorescence (IF), immunohistochemistry (IHC), immunoprecipitation (IP), or integrin activity assay (IA): α-Actinin (BM75.2, mouse anti-human, Abcam; 1:1000 WB), α 1 -integrin (TS2/7, mouse anti-human/anti-mouse, Abcam; 1:50 IF), α 2 -integrin (6 F1, mouse anti-human/anti-mouse, DSHB; 1:60 IF), α 3 -integrin (P1B5, mouse anti-human/anti-mouse, DSHB; 1:60 IF), α 5 -integrin (BIIG2, rat anti-human/anti-mouse, DSHB; 1:10 IF), α v -integrin (PE-P2 W7 mouse anti-human/anti-mouse, sc-9969; IF 1:300), β 1 -integrin (HMβ1-1, armenian hamster anti-mouse, Bio Legend; 1:300 IF; AIIB2, rat anti-human/anti-mouse, DSHB; 1:600 IF, IA; M-106, rabbit anti-mouse/anti-human, Santa Cruz; 1:500 WB; D2E5, rabbit anti-human, Cell Signaling; 1:1000 WB), human β 1 -integrin (P5D2, mouse anti-human, DSHB, 2.5 μg IP; 9EG7, rat anti- human, DSHB 2.5 μg IP; AIIB2, rat anti-human, DSHB; 2.5 μg IP), β 3 -integrin (2 C9.G3, arm. hamster anti-mouse, eBioscience; 1:300 IF; PM6/13, mouse anti-human, Abcam; 1:100 IF), β 5 -integrin (KN-52, mouse anti-mouse/human, eBioscience; IF 1:300), Focal adhesion kinase (FAK) (77, mouse anti-human, BD; 1:250 WB), integrin-linked kinase (ILK) (EP1593Y, rabbit anti-human, Epitomics; 1:800 WB), Kindlin-2 (3 A3, mouse anti-human, Millipore; 1:200 WB, 1:250 IF), Laminin (ab11575, rabbit anti-mouse, Abcam; 1:300 IHC), Nestin (rat-401, anti-mouse, Millipore; IHC 1:200), Paxillin (5H11, mouse monoclonal, Thermo Scientific; 1:1000 WB), hPPM1F (17,020–1-AP, rabbit anti-human, Protein-Tech; 1:1000 WB), mPPM1F (#1147, rabbit anti-mouse PPM1F; generated at the central animal care facility; University of Konstanz; 1:200 WB; see Additional File2: Fig. S2), FilaminA (EP2405Y, IgG, rabbit anti-human, Epitomics; 1:125.000 WB), Tubulin (E7, IgG1, mouse anti-human, DSHB; 1:1000), Talin (8 d4, mouse anti-human, Thermo Scientific; 1:800 WB, 1:40 IF), Vinculin (hVIN-1, mouse anti-human, Sigma; 1:2000 WB, 1:200 IF), Zyxin (Zol301, mouse anti-human, Abcam; 1:1000 WB), Dylight488-conjugated goat anti-mouse IgG (Jackson; 1:200), Cy3-conjugated goat anti-rabbit IgG (Jackson; 1:200), Cy3-conjugated goat anti-mouse IgG (Jackson; 1:200), Cy5-conjugated goat anti-mouse IgG (Jackson; 1:200), RhodamineRed-conjugated goat anti-rat IgG (Jackson; 1:200), RhodamineRed-conjugated goat anti-Armenian Hamster IgG (Jackson; 1:200), HRP-conjugated goat anti-mouse IgG (Jackson; WB 1:10 000), HRP-conjugated goat anti-rat IgG (Santa Cruz; 1:250), HRP-conjugated goat anti-rabbit IgG (Jackson; WB 1:3000), unspecific control IgG (anti-mouse, 96/1, generated at the Tierforschungsanlage; University of Konstanz; anti-rat, MJ7/18 Endoglin, DSHB).

Techniques: Activity Assay, Control, Western Blot, Staining, Confocal Microscopy, Expressing, Suspension

Journal: bioRxiv

Article Title: Development of Integrin α5β1-targeted PET/NIR imaging probes for glioblastoma intraoperative navigation and intracavity targeted radionuclide therapy

doi: 10.64898/2026.01.09.698741

Figure Lengend Snippet: (A) Strategies for modification of GS, including amino acid mutation, covalent modification, cyclization, and multivalency. (B) SPR sensorgrams demonstrating the binding affinity of GS and GR for human integrin α5β1 in a concentration-dependent manner. The equilibrium dissociation constant (K D ) of each peptide was calculated based on SPR measurements. The K D values of each precursor are shown. (C) Molecular docking of GR and GS binding integrin a5b1 protein (grey; PDB: 7NWL) showing the selected possible ligation residues. (D-F) Analysis of integrin α5β1 expression in U87MG cells and tumor tissues by western blot ( D ), and immunohistochemistry ( E-F ) analysis. The band for integrin α5 was approximately 150 kDa. M, marker. C, cell. T, tumor. For immunofluorescence images, green is for integrin α5, red for integrin β1, and blue for nucleus. Scale bar, 50□μm ( E-F ). (G) In vitro cellular uptake of [ 68 Ga]GS and [ 68 Ga]GR in U87MG cell lines. All results are expressed as means ± SEM, as indicated in at least three independent experiments. “*” represents differences compared with the [ 68 Ga]GS. * p < 0.05.

Article Snippet: Recombinant human integrin α5β1 (alpha 5 beta 1, HY-P77718, MCE, NJ, USA) was immobilized on a CM5 sensor chip at 25 □ following a standard amine coupling kit.

Techniques: Modification, Mutagenesis, Binding Assay, Concentration Assay, Ligation, Expressing, Western Blot, Immunohistochemistry, Marker, Immunofluorescence, In Vitro

Journal: bioRxiv

Article Title: Development of Integrin α5β1-targeted PET/NIR imaging probes for glioblastoma intraoperative navigation and intracavity targeted radionuclide therapy

doi: 10.64898/2026.01.09.698741

Figure Lengend Snippet: (A) Immunofluorescence images show the expression of integrin α5β1, GFAP, and NEUN of U87MG tumors in orthotopic glioblastoma tumor-bearing mice. Green indicates integrin α5, Red for NEUN, yellow for GFAP, and blue for nucleus. Scale bar, 1 mm. (B-C) Representative fluorescence imaging of mice bearing in orthotopic U87MG tumors ( B ) and ex vivo brains ( C ). Cy5-GS and Cy5-GR were intravenously injected with a dose of 5 mg/kg. (D) Quantification of fluorescence intensity in tumors corresponding to ( C ). (E) Immunohistochemical analysis of integrin α5β1 expression in brain tissue sections from orthotopic glioblastoma tumor-bearing mice. Scale bar, 1 mm. (F-G) Immunofluorescence images of brain tissue sections from orthotopic glioblastoma tumor-bearing mice treated with Cy5-GS ( F ) and Cy5-GR ( G ). Green indicates integrin α5, Red for GS or GR, and blue for nucleus. Scale bar, 1 mm. (H-I) Magnific imaging of brain tissue sections from orthotopic glioblastoma tumor-bearing mice treated with GS-Cy5. Scale bar, 50 μm. All results are expressed as means ± SEM, as indicated in at least three independent experiments. A multiple t-test was used when two groups were compared. The symbol “*” represents differences compared with the Cy5-GS. *** p < 0.001.

Article Snippet: Recombinant human integrin α5β1 (alpha 5 beta 1, HY-P77718, MCE, NJ, USA) was immobilized on a CM5 sensor chip at 25 □ following a standard amine coupling kit.

Techniques: Immunofluorescence, Expressing, Fluorescence, Imaging, Ex Vivo, Injection, Immunohistochemical staining

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