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α 5 integrin (Developmental Studies Hybridoma Bank)

Name: α 5 integrin
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Rating: 94 (76 reviews)

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Developmental Studies Hybridoma Bank α 5 integrin

Increased <t>integrin</t> 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

α 5 Integrin, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 94/100, based on 76 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "The phosphatase PPM1F, a negative regulator of integrin activity, is essential for embryonic development and controls tumor cell invasion"

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

Journal: BMC Biology

doi: 10.1186/s12915-025-02254-3

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

Figure Legend 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

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

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 )

Figure Legend 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 )

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

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

Figure Legend 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

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

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

Figure Legend 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

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

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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 )

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

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

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

MFAP4 binding and activation of RGD-dependent integrins (A) MFAP4 promotes adhesion of human pulmonary microvascular endothelial cells (HPMECs) in a dose-dependent manner. (B) MFAP4-mediated adhesion of HPMEC can be inhibited by RGD-containing peptide (GRGDS) but not control (SDGRG) peptide. Data are means (SD) of n = 3 independent experiments. Significance is calculated by two-way ANOVA. MFI, mean fluorescence intensity. Immobilized recombinant MFAP4 (2 μg/mL) was incubated with increasing concentrations of recombinant integrins. Integrin binding to recombinant MFAP4 was detected for (C) integrins α V β 3/5/6 and αIIbβ 3 but not (D) integrins α V β 1/8 and α 4/5/8 β 1 . Used detection antibodies are shown in brackets. Data are shown as means (SD) of n = 3 independent experiments. Relative band density of (E) phosphorylated FAK (pFAK)/total FAK and (F) pERK/total ERK in HPMEC after cellular adhesion to poly-D-lysine (PDL, negative control), vitronectin (VTN, positive control), or MFAP4 are shown in representative western blots and quantitated mean (SD) of n = 3 independent experiments. Quantifications of western blotting was analyzed by two-way ANOVA for MFAP4 relative to negative control and the significance is provided for treatment factor only (independent of the significant time factor).

Journal: Molecular Therapy

Article Title: Pharmacological blocking of microfibrillar-associated protein 4 reduces retinal neoangiogenesis and vascular leakage

doi: 10.1016/j.ymthe.2025.01.038

Figure Lengend Snippet: MFAP4 binding and activation of RGD-dependent integrins (A) MFAP4 promotes adhesion of human pulmonary microvascular endothelial cells (HPMECs) in a dose-dependent manner. (B) MFAP4-mediated adhesion of HPMEC can be inhibited by RGD-containing peptide (GRGDS) but not control (SDGRG) peptide. Data are means (SD) of n = 3 independent experiments. Significance is calculated by two-way ANOVA. MFI, mean fluorescence intensity. Immobilized recombinant MFAP4 (2 μg/mL) was incubated with increasing concentrations of recombinant integrins. Integrin binding to recombinant MFAP4 was detected for (C) integrins α V β 3/5/6 and αIIbβ 3 but not (D) integrins α V β 1/8 and α 4/5/8 β 1 . Used detection antibodies are shown in brackets. Data are shown as means (SD) of n = 3 independent experiments. Relative band density of (E) phosphorylated FAK (pFAK)/total FAK and (F) pERK/total ERK in HPMEC after cellular adhesion to poly-D-lysine (PDL, negative control), vitronectin (VTN, positive control), or MFAP4 are shown in representative western blots and quantitated mean (SD) of n = 3 independent experiments. Quantifications of western blotting was analyzed by two-way ANOVA for MFAP4 relative to negative control and the significance is provided for treatment factor only (independent of the significant time factor).

Article Snippet: HPMECs were detached with Accutase (Fisher Scientific) and suspended at 200,000 cells/sample in FACS buffer containing 10 μg/mL mouse mAbs against integrin α V β 3 (Millipore, cat. # MAB1976), integrin α V β 5 (Santa Cruz, cat. # sc-13588), with anti-ovalbumin (HYB099-01), The State Serum Institute) as isotype control (IC) for 1 h at 4°C.

Techniques: Binding Assay, Activation Assay, Control, Fluorescence, Recombinant, Incubation, Negative Control, Positive Control, Western Blot

The AS0326 antibody blocks MFAP4s interaction with endothelial integrins (A) HPMECs were subjected to MFAP4-mediated adhesion, and inhibition of adhesion was tested with integrin α V β 3 - or integrin α V β 5 -blocking antibodies. (B) mAS0326 antibody (mAS) blocks HPMEC adhesion to MFAP4. MFI is of fluorescently labeled cells. IC, isotype control. Significance is calculated relative to the MFAP4-treated positive control in (A) and (B). Flow cytometry staining of (C) integrin α V β 3 and (D) integrin α V β 5 in HPMEC compared to IC. Representative histograms of n = 3 independent experiments are shown. Human primary retinal endothelial cells (RECs) were seeded on immobilized albumin or MFAP4. RECs were stimulated with VEGF and 24-h proliferation was assessed. (E) REC proliferation was co-treated with integrin α V β 3 - or α V β 5 -blocking antibodies or hAS0326 (hAS). REC migration for 3.5 h on MFAP4-coated surface was assessed by Transwell assay using VEGF as chemoattractant. (F) MFAP4-dependent migration was treated with integrin α V β 3 - or α V β 5 -blocking antibodies or hAS0326. Data are shown as individual datapoints with mean (SD) of n = 3 independent experiments. Data are normalized to albumin control. Significance is calculated relative to the hAS0326 treatment group for (E) and (F). Significance calculations are performed using one-way ANOVA followed by Dunnett’s multiple comparison test. Representative images of REC’s (purple) migrated through the pores of the Transwell assay inserts when migration on (G) albumin-coated insert or (H) MFAP4-coated insert. Bar, 100 μm. All antibodies were provided in 10 μg/mL doses for (A), (B), (E), and (F).

Journal: Molecular Therapy

Article Title: Pharmacological blocking of microfibrillar-associated protein 4 reduces retinal neoangiogenesis and vascular leakage

doi: 10.1016/j.ymthe.2025.01.038

Figure Lengend Snippet: The AS0326 antibody blocks MFAP4s interaction with endothelial integrins (A) HPMECs were subjected to MFAP4-mediated adhesion, and inhibition of adhesion was tested with integrin α V β 3 - or integrin α V β 5 -blocking antibodies. (B) mAS0326 antibody (mAS) blocks HPMEC adhesion to MFAP4. MFI is of fluorescently labeled cells. IC, isotype control. Significance is calculated relative to the MFAP4-treated positive control in (A) and (B). Flow cytometry staining of (C) integrin α V β 3 and (D) integrin α V β 5 in HPMEC compared to IC. Representative histograms of n = 3 independent experiments are shown. Human primary retinal endothelial cells (RECs) were seeded on immobilized albumin or MFAP4. RECs were stimulated with VEGF and 24-h proliferation was assessed. (E) REC proliferation was co-treated with integrin α V β 3 - or α V β 5 -blocking antibodies or hAS0326 (hAS). REC migration for 3.5 h on MFAP4-coated surface was assessed by Transwell assay using VEGF as chemoattractant. (F) MFAP4-dependent migration was treated with integrin α V β 3 - or α V β 5 -blocking antibodies or hAS0326. Data are shown as individual datapoints with mean (SD) of n = 3 independent experiments. Data are normalized to albumin control. Significance is calculated relative to the hAS0326 treatment group for (E) and (F). Significance calculations are performed using one-way ANOVA followed by Dunnett’s multiple comparison test. Representative images of REC’s (purple) migrated through the pores of the Transwell assay inserts when migration on (G) albumin-coated insert or (H) MFAP4-coated insert. Bar, 100 μm. All antibodies were provided in 10 μg/mL doses for (A), (B), (E), and (F).

Techniques: Inhibition, Blocking Assay, Labeling, Control, Positive Control, Flow Cytometry, Staining, Migration, Transwell Assay, Comparison

Specificity of the AS0326 antibody (A) mAS0326 and (B) hAS0326 efficiently detect MFAP4 in WT ( Mfap4 +/+ ) mouse serum, but not in MFAP4-deficient ( Mfap4 −/− ) mouse serum. (C) Increasing concentrations of hAS0326-Fab or hAS0326Y94A L-CDR3 Fab variant were applied for inhibition of 1.5 nM MFAP4 binding to an excess of immobilized integrin α V β 3 . (D–F) Brown symbols, HG-HYB7-5; purple symbols, mAS0326; red symbols; hAS0326. (D) Both mAS0326 and hAS0326 efficiently detect immobilized recombinant human MFAP4 (rhMFAP4 coating) variant carrying RGD-AAA mutation (RGD-AAA), while HG-HYB7-5 antibody show RGD-dependent binding. Both mAS0326 and hAS0326 efficiently detect immobilized (E) recombinant mouse (rm) MFAP4, and (F) rhMFAP4, while HG-HYB7-5 is rhMFAP4 specific. Data are shown as means (SD) of n = 3 independent experiments.

Journal: Molecular Therapy

Article Title: Pharmacological blocking of microfibrillar-associated protein 4 reduces retinal neoangiogenesis and vascular leakage

doi: 10.1016/j.ymthe.2025.01.038

Figure Lengend Snippet: Specificity of the AS0326 antibody (A) mAS0326 and (B) hAS0326 efficiently detect MFAP4 in WT ( Mfap4 +/+ ) mouse serum, but not in MFAP4-deficient ( Mfap4 −/− ) mouse serum. (C) Increasing concentrations of hAS0326-Fab or hAS0326Y94A L-CDR3 Fab variant were applied for inhibition of 1.5 nM MFAP4 binding to an excess of immobilized integrin α V β 3 . (D–F) Brown symbols, HG-HYB7-5; purple symbols, mAS0326; red symbols; hAS0326. (D) Both mAS0326 and hAS0326 efficiently detect immobilized recombinant human MFAP4 (rhMFAP4 coating) variant carrying RGD-AAA mutation (RGD-AAA), while HG-HYB7-5 antibody show RGD-dependent binding. Both mAS0326 and hAS0326 efficiently detect immobilized (E) recombinant mouse (rm) MFAP4, and (F) rhMFAP4, while HG-HYB7-5 is rhMFAP4 specific. Data are shown as means (SD) of n = 3 independent experiments.

Techniques: Variant Assay, Inhibition, Binding Assay, Recombinant, Mutagenesis

Structural basis for the hAS0326 Fab interaction with MFAP4 The hAS0326 Fab complex and the MFAP4 octamer. (A) Cartoon representation of the octameric MFAP4-Fab complex. (B) Each Fab contacts a single MFAP4 monomer at an epitope next to the MFAP4 N terminus but far from the binding site for the Ca 2+ ion (cyan sphere). (C) The MFAP4 octamer is built from two FIBCD1-like tetramers (colored orange and red) with eight Ca 2+ ions located at the tetramer-tetramer interface. (D) Inside view showing the concave face of the tetramer. (E) Top view of the convex face of the tetramer with the four bound Fab molecules. Orange spheres mark the first MFAP4 residue that can be located. Presumably, the disordered RGD integrin-binding motif at the N terminus of MFAP4 is trapped and inaccessible inside the funnel-shaped space formed by the Fab molecules. Nt, N terminus. (F) The epitope mapped on MFAP4. Residues primarily in contact with heavy-chain and light-chain CDRs are colored blue and green, respectively. (G) Details of the intermolecular interaction centered on MFAP4 residues 24–29. Putative polar interactions are indicated by dotted lines.

Journal: Molecular Therapy

Article Title: Pharmacological blocking of microfibrillar-associated protein 4 reduces retinal neoangiogenesis and vascular leakage

doi: 10.1016/j.ymthe.2025.01.038

Figure Lengend Snippet: Structural basis for the hAS0326 Fab interaction with MFAP4 The hAS0326 Fab complex and the MFAP4 octamer. (A) Cartoon representation of the octameric MFAP4-Fab complex. (B) Each Fab contacts a single MFAP4 monomer at an epitope next to the MFAP4 N terminus but far from the binding site for the Ca 2+ ion (cyan sphere). (C) The MFAP4 octamer is built from two FIBCD1-like tetramers (colored orange and red) with eight Ca 2+ ions located at the tetramer-tetramer interface. (D) Inside view showing the concave face of the tetramer. (E) Top view of the convex face of the tetramer with the four bound Fab molecules. Orange spheres mark the first MFAP4 residue that can be located. Presumably, the disordered RGD integrin-binding motif at the N terminus of MFAP4 is trapped and inaccessible inside the funnel-shaped space formed by the Fab molecules. Nt, N terminus. (F) The epitope mapped on MFAP4. Residues primarily in contact with heavy-chain and light-chain CDRs are colored blue and green, respectively. (G) Details of the intermolecular interaction centered on MFAP4 residues 24–29. Putative polar interactions are indicated by dotted lines.

Techniques: Binding Assay, Residue

The macular proteome of the DL-AAA-induced model of chronic retinopathy supports integrin involvement Macular punches including choroid, RPE, and retina obtained at end-study (week 22) were analyzed by mass spectrometry and following analysis using clusterProfiler 4.0 package in R. The proteomes were generated from n = 3 eyes for non-diseased control, n = 5 eyes for DL-AAA treatment, and n = 5 eyes for hAS0326 treatment. (A) Over-representation analysis (ORA) showing the effect of DL-AAA treatment (relative to no DL-AAA treatment) and hAS0326 treatment of DL-AAA-treated eyes (relative to DL-AAA treatment), respectively. The plot was generated using compareCluster function with default settings. All ontologies with significant regulation post correction for multiple testing using the Benjamini-Hochberg procedure are shown. Dot sizes indicate the ratio (i.e., the coverage of a given term by proteins regulated for each comparison), and dot colors indicate the level of significance. (B–E) Volcano plots showing regulation of detected proteins underlying selected GO terms. For selected, significantly regulated gene ontologies, the ORA input proteins displaying significant regulation are highlighted in color. Protein IDs are shown for the top three proteins with lowest p belonging to a particular ontology. Moreover, protein IDs are shown for specific proteins of interest.

Journal: Molecular Therapy

Article Title: Pharmacological blocking of microfibrillar-associated protein 4 reduces retinal neoangiogenesis and vascular leakage

doi: 10.1016/j.ymthe.2025.01.038

Figure Lengend Snippet: The macular proteome of the DL-AAA-induced model of chronic retinopathy supports integrin involvement Macular punches including choroid, RPE, and retina obtained at end-study (week 22) were analyzed by mass spectrometry and following analysis using clusterProfiler 4.0 package in R. The proteomes were generated from n = 3 eyes for non-diseased control, n = 5 eyes for DL-AAA treatment, and n = 5 eyes for hAS0326 treatment. (A) Over-representation analysis (ORA) showing the effect of DL-AAA treatment (relative to no DL-AAA treatment) and hAS0326 treatment of DL-AAA-treated eyes (relative to DL-AAA treatment), respectively. The plot was generated using compareCluster function with default settings. All ontologies with significant regulation post correction for multiple testing using the Benjamini-Hochberg procedure are shown. Dot sizes indicate the ratio (i.e., the coverage of a given term by proteins regulated for each comparison), and dot colors indicate the level of significance. (B–E) Volcano plots showing regulation of detected proteins underlying selected GO terms. For selected, significantly regulated gene ontologies, the ORA input proteins displaying significant regulation are highlighted in color. Protein IDs are shown for the top three proteins with lowest p belonging to a particular ontology. Moreover, protein IDs are shown for specific proteins of interest.

Techniques: Mass Spectrometry, Generated, Control, Comparison

In vitro binding affinity and stability studies of [ 64 Cu]QM-2301, [ 64 Cu]QM-2302, and [ 64 Cu]QM-2303. (A) SPR sensorgrams demonstrating the binding affinity of QM-2301, QM-2302, and QM-2303 for human integrin α 5 β 1 in a concentration-dependent manner. (B) The equilibrium dissociation constant ( Κ D ) of each peptide was calculated based on SPR measurements. The K D values of each precursor are shown. (C, D) Schematic diagram of the binding patterns of [ 64 Cu]QM-2301, [ 64 Cu]QM-2302 (C), and [ 64 Cu]QM-2303 (D) to integrin α 5 β 1. Monomeric [ 64 Cu]QM-2301, and [ 64 Cu]QM-2302 bind to receptors in a single-network pattern. For [ 64 Cu]QM-2303, the PEGibody-based radiotracer, one PEGibody can bind to more than two integrin α 5 β 1 receptors, exhibiting better binding affinity. (E, F) The expression of integrin α 5 β 1 in B16F10 cells was analyzed by flow cytometry (E) and Western blotting (F). For flow cytometry assays, an anti-integrin α 5 + β 1 antibody was used. For Western blot assays, integrin α 5 (∼150 kDa) and integrin β 1 (∼138 kDa) were examined using two antibodies. (G) The stability of [ 64 Cu]QM-2301, [ 64 Cu]QM-2302, and [ 64 Cu]QM-2303 after coincubation with mouse serum within 1 h, as indicated by radio-HPLC. (H) I n vitro cell uptake of [ 64 Cu]QM-2301, [ 64 Cu]QM-2302, and [ 64 Cu]QM-2303 (750 KBq/mL) when incubated with B16F10 cells for different time period. (I) IC 50 of [ 64 Cu]QM-2301, [ 64 Cu]QM-2302, and [ 64 Cu]QM-2303 when inhibited with antibodies at different concentrations. ∗ P < 0.05, ∗∗ P < 0.01. All the quantitative experiments were performed independently at least three times (data are the mean ± SD, n = 3).

Journal: Acta Pharmaceutica Sinica. B

Article Title: Enhanced radiotheranostic targeting of integrin α 5 β 1 with PEGylation-enabled peptide multidisplay platform (PEGibody): A strategy for prolonged tumor retention with fast blood clearance

doi: 10.1016/j.apsb.2024.07.006

Figure Lengend Snippet: In vitro binding affinity and stability studies of [ 64 Cu]QM-2301, [ 64 Cu]QM-2302, and [ 64 Cu]QM-2303. (A) SPR sensorgrams demonstrating the binding affinity of QM-2301, QM-2302, and QM-2303 for human integrin α 5 β 1 in a concentration-dependent manner. (B) The equilibrium dissociation constant ( Κ D ) of each peptide was calculated based on SPR measurements. The K D values of each precursor are shown. (C, D) Schematic diagram of the binding patterns of [ 64 Cu]QM-2301, [ 64 Cu]QM-2302 (C), and [ 64 Cu]QM-2303 (D) to integrin α 5 β 1. Monomeric [ 64 Cu]QM-2301, and [ 64 Cu]QM-2302 bind to receptors in a single-network pattern. For [ 64 Cu]QM-2303, the PEGibody-based radiotracer, one PEGibody can bind to more than two integrin α 5 β 1 receptors, exhibiting better binding affinity. (E, F) The expression of integrin α 5 β 1 in B16F10 cells was analyzed by flow cytometry (E) and Western blotting (F). For flow cytometry assays, an anti-integrin α 5 + β 1 antibody was used. For Western blot assays, integrin α 5 (∼150 kDa) and integrin β 1 (∼138 kDa) were examined using two antibodies. (G) The stability of [ 64 Cu]QM-2301, [ 64 Cu]QM-2302, and [ 64 Cu]QM-2303 after coincubation with mouse serum within 1 h, as indicated by radio-HPLC. (H) I n vitro cell uptake of [ 64 Cu]QM-2301, [ 64 Cu]QM-2302, and [ 64 Cu]QM-2303 (750 KBq/mL) when incubated with B16F10 cells for different time period. (I) IC 50 of [ 64 Cu]QM-2301, [ 64 Cu]QM-2302, and [ 64 Cu]QM-2303 when inhibited with antibodies at different concentrations. ∗ P < 0.05, ∗∗ P < 0.01. All the quantitative experiments were performed independently at least three times (data are the mean ± SD, n = 3).

Article Snippet: Recombinant human integrin α 5 β 1 (alpha 5 beta 1, HY-P77718, MCE, NJ, USA) was immobilized on a CM5 sensor chip using a standard amine coupling kit at a temperature of 25 °C.

Techniques: In Vitro, Binding Assay, Concentration Assay, Expressing, Flow Cytometry, Western Blot, Incubation

A-C: Heat maps of gene expression of boron transporter ( NaBC1 ), myogenesis markers ( MYOD, MYOGENIN ), AKT/mTOR pathway ( AKT, mTOR ), muscle metabolism ( IL-GFR , INSR , GDF11 , VEGFR ), cell adhesion-related genes ( ALPHAV , ALPHA5 , ALPHA7 , BETA1 , BETA3 , BETA5 Integrins) in C2C12 myoblasts seeded on PAAm hydrogels of different stiffnesses, functionalized with fibronectin (FN) and stimulated with soluble boron (B) (at 0.59 and 1.47 mM) for 4 (A), 8 (B) or 24 hours (C) compared to untreated cells on cell culture plates, as measured by qPCR. D: Heat map of gene expression in C2C12 myoblasts seeded on PAAm hydrogels of different stiffnesses, functionalized with FN and stimulated with soluble B (at 0.59 and 1.47 mM) for 96 hours in myogenic differentiation conditions, as measured by qPCR. For figures A-D, n = 3 biological replicates with 3 technical replicates. E: Colocalization assays were performed by using the Duolink ® PLA protein detection technology, which is based on in situ proximity ligation assay (PLA) that allows the visualization and quantification of protein-protein interactions when proteins are present within 40 nm. Representative images showing the colocalization dots of NaBC1/α 5 , NaBC1/α v and NaBC1/β 4 in C2C12 myoblasts seeded on rigid PAAm hydrogels, functionalized with FN for 1 hour and stimulated with soluble B (0.59 and 1.47 mM). Magenta: colocalization dots; Cyan: DAPI. Scale bar: 50 µm. F: Quantification of number of colocalization dots of NaBC1/α 5 , NaBC1/α v and NaBC1/β 4 . n = 30 cells from 3 different biological replicates. Data are represented as Mean ± Standard Deviation, and differences are considered significant for p ≤ 0.05 using one-way ANOVAs (Tukey’s multiple comparisons tests) for multiple comparisons. *p ≤ 0.05

Journal: bioRxiv

Article Title: NaBC1 boron transporter enables myoblast response to substrate rigidity via fibronectin-binding integrins

doi: 10.1101/2024.06.14.599051

Figure Lengend Snippet: A-C: Heat maps of gene expression of boron transporter ( NaBC1 ), myogenesis markers ( MYOD, MYOGENIN ), AKT/mTOR pathway ( AKT, mTOR ), muscle metabolism ( IL-GFR , INSR , GDF11 , VEGFR ), cell adhesion-related genes ( ALPHAV , ALPHA5 , ALPHA7 , BETA1 , BETA3 , BETA5 Integrins) in C2C12 myoblasts seeded on PAAm hydrogels of different stiffnesses, functionalized with fibronectin (FN) and stimulated with soluble boron (B) (at 0.59 and 1.47 mM) for 4 (A), 8 (B) or 24 hours (C) compared to untreated cells on cell culture plates, as measured by qPCR. D: Heat map of gene expression in C2C12 myoblasts seeded on PAAm hydrogels of different stiffnesses, functionalized with FN and stimulated with soluble B (at 0.59 and 1.47 mM) for 96 hours in myogenic differentiation conditions, as measured by qPCR. For figures A-D, n = 3 biological replicates with 3 technical replicates. E: Colocalization assays were performed by using the Duolink ® PLA protein detection technology, which is based on in situ proximity ligation assay (PLA) that allows the visualization and quantification of protein-protein interactions when proteins are present within 40 nm. Representative images showing the colocalization dots of NaBC1/α 5 , NaBC1/α v and NaBC1/β 4 in C2C12 myoblasts seeded on rigid PAAm hydrogels, functionalized with FN for 1 hour and stimulated with soluble B (0.59 and 1.47 mM). Magenta: colocalization dots; Cyan: DAPI. Scale bar: 50 µm. F: Quantification of number of colocalization dots of NaBC1/α 5 , NaBC1/α v and NaBC1/β 4 . n = 30 cells from 3 different biological replicates. Data are represented as Mean ± Standard Deviation, and differences are considered significant for p ≤ 0.05 using one-way ANOVAs (Tukey’s multiple comparisons tests) for multiple comparisons. *p ≤ 0.05

Article Snippet: Specific primary antibodies used were: anti-NaBC1 (Invitrogen, 1:200), anti-integrin α v (Abcam, 1:500), anti-integrin α 5 (Abcam, 1:500) and anti-integrin β (Abcam, 1:500).

Techniques: Expressing, Cell Culture, In Situ, Proximity Ligation Assay, Standard Deviation

The results reported in panels A-J derive from experiments in which C2C12 myoblasts were seeded on PAAm hydrogels of different stiffnesses (soft, medium, and rigid) that were functionalized with laminin-111 and stimulated with soluble boron ions at two different concentrations (0.59 and 1.47 mM). A: Representative immunofluorescence images of C2C12 myoblasts cultured as described. Magenta: actin cytoskeleton; Cyan: DAPI. Scale bar: 20 µm. B: Quantification of cell area of C2C12 myoblasts cultured as described. n = 10 cells from 3 different biological replicates. C: Representative immunofluorescence images of C2C12 myoblasts cultured as described. Magenta: vinculin. Scale bar: 20 µm. D: Quantification of focal adhesion (FA) length in C2C12 myoblasts cultured as described. n = 10 cells from 3 different biological replicates. E: Quantification of actin retrograde flow in C2C12 myoblasts cultured as described. n = 5 cells with at least 5 different flow areas per cell. F: Quantification of Brillouin shift in C2C12 myoblasts cultured as described and imaged with Brillouin microscopy. n = 10 cells from 3 different biological replicates. G: Quantification of cell stiffness by nanoindentation of C2C12 myoblasts seeded on glass coverslips functionalized with laminin-111 and stimulated with soluble B, as described. n = 10 cells with 9 indentations on each single cell from 3 different biological replicates. H: Representative traction maps of C2C12 myoblasts cultured as described. I: Quantification of traction forces exerted by C2C12 myoblasts when cultured as described. n = 30 cells from 10 different locations within each hydrogel from 3 different biological replicates. J: Colocalization assays were performed by using the Duolink ® PLA protein detection technology, which is based on in situ proximity ligation assay (PLA) that allows the visualization and quantification of protein-protein interactions when proteins are present within 40 nm. Representative images of colocalization dots of NaBC1/α 5 , NaBC1/α v and NaBC1/β 4 in C2C12 myoblasts cultured as described. Magenta: colocalization dots; Cyan: DAPI. Scale bar: 50 µm. K: Quantification of number of colocalization dots of NaBC1/α 5 , NaBC1/α v and NaBC1/β 4 . n = 30 cells from 3 different biological replicates. Data are represented as Mean ± Standard Deviation, and differences are considered significant for p ≤ 0.05 using one-way ANOVAs or two-way ANOVAs (Tukey’s multiple comparisons tests) for multiple comparisons. *p ≤ 0.05, ***p ≤ 0.001, ****p ≤ 0.0001

Journal: bioRxiv

Article Title: NaBC1 boron transporter enables myoblast response to substrate rigidity via fibronectin-binding integrins

doi: 10.1101/2024.06.14.599051

Figure Lengend Snippet: The results reported in panels A-J derive from experiments in which C2C12 myoblasts were seeded on PAAm hydrogels of different stiffnesses (soft, medium, and rigid) that were functionalized with laminin-111 and stimulated with soluble boron ions at two different concentrations (0.59 and 1.47 mM). A: Representative immunofluorescence images of C2C12 myoblasts cultured as described. Magenta: actin cytoskeleton; Cyan: DAPI. Scale bar: 20 µm. B: Quantification of cell area of C2C12 myoblasts cultured as described. n = 10 cells from 3 different biological replicates. C: Representative immunofluorescence images of C2C12 myoblasts cultured as described. Magenta: vinculin. Scale bar: 20 µm. D: Quantification of focal adhesion (FA) length in C2C12 myoblasts cultured as described. n = 10 cells from 3 different biological replicates. E: Quantification of actin retrograde flow in C2C12 myoblasts cultured as described. n = 5 cells with at least 5 different flow areas per cell. F: Quantification of Brillouin shift in C2C12 myoblasts cultured as described and imaged with Brillouin microscopy. n = 10 cells from 3 different biological replicates. G: Quantification of cell stiffness by nanoindentation of C2C12 myoblasts seeded on glass coverslips functionalized with laminin-111 and stimulated with soluble B, as described. n = 10 cells with 9 indentations on each single cell from 3 different biological replicates. H: Representative traction maps of C2C12 myoblasts cultured as described. I: Quantification of traction forces exerted by C2C12 myoblasts when cultured as described. n = 30 cells from 10 different locations within each hydrogel from 3 different biological replicates. J: Colocalization assays were performed by using the Duolink ® PLA protein detection technology, which is based on in situ proximity ligation assay (PLA) that allows the visualization and quantification of protein-protein interactions when proteins are present within 40 nm. Representative images of colocalization dots of NaBC1/α 5 , NaBC1/α v and NaBC1/β 4 in C2C12 myoblasts cultured as described. Magenta: colocalization dots; Cyan: DAPI. Scale bar: 50 µm. K: Quantification of number of colocalization dots of NaBC1/α 5 , NaBC1/α v and NaBC1/β 4 . n = 30 cells from 3 different biological replicates. Data are represented as Mean ± Standard Deviation, and differences are considered significant for p ≤ 0.05 using one-way ANOVAs or two-way ANOVAs (Tukey’s multiple comparisons tests) for multiple comparisons. *p ≤ 0.05, ***p ≤ 0.001, ****p ≤ 0.0001

Article Snippet: Specific primary antibodies used were: anti-NaBC1 (Invitrogen, 1:200), anti-integrin α v (Abcam, 1:500), anti-integrin α 5 (Abcam, 1:500) and anti-integrin β (Abcam, 1:500).

Techniques: Immunofluorescence, Cell Culture, Microscopy, In Situ, Proximity Ligation Assay, Standard Deviation

ZIKV-LAV entry in human GBM cells is mediated by Axl and integrin α v β 5 . Evaluation of the effect of siRNA-mediated knockdown of either Axl or integrin α v β 5 gene on A – B viral entry in DBTRG ( A ) and T98G ( B ) cells; C – D protein expression of Axl ( C ) and integrin α v β 5 ( D ) on the cell surface; and E – F intracellular viral replication in DBTRG ( E ) and T98G ( F ) cells. SCR , scrambled siRNA. Int.α v β 5 , integrin α v β 5. Data are presented as mean ± SEM. Non-parametric Mann–Whitney test or Kruskal–Wallis test with Dunn’s post-hoc correction was used to compare groups. p -values are shown accordingly: * p < 0.05. ** p < 0.005, *** p < 0.001

Journal: Journal of Translational Medicine

Article Title: Repurposing of Zika virus live-attenuated vaccine (ZIKV-LAV) strains as oncolytic viruses targeting human glioblastoma multiforme cells

doi: 10.1186/s12967-024-04930-4

Figure Lengend Snippet: ZIKV-LAV entry in human GBM cells is mediated by Axl and integrin α v β 5 . Evaluation of the effect of siRNA-mediated knockdown of either Axl or integrin α v β 5 gene on A – B viral entry in DBTRG ( A ) and T98G ( B ) cells; C – D protein expression of Axl ( C ) and integrin α v β 5 ( D ) on the cell surface; and E – F intracellular viral replication in DBTRG ( E ) and T98G ( F ) cells. SCR , scrambled siRNA. Int.α v β 5 , integrin α v β 5. Data are presented as mean ± SEM. Non-parametric Mann–Whitney test or Kruskal–Wallis test with Dunn’s post-hoc correction was used to compare groups. p -values are shown accordingly: * p < 0.05. ** p < 0.005, *** p < 0.001

Article Snippet: The Human Protein Atlas report that Axl and integrin β 5 are highly expressed in neuroprogenitor cells (NPC) but not in terminally differentiated neurons [ , ], indicating that the GBM selectivity of ZIKV-LAV infection is partly explained by differential Axl and integrin α v β 5 expression.

Techniques: Knockdown, Expressing, MANN-WHITNEY

Proposed model of human GBM cell death mediated by ZIKV-LAV infection. A – D General virus infection life cycle. A ZIKV-LAV enters human GBM cells through Axl and integrin α v β 5 cellular receptors. B The ZIKV-LAV genome is translated to express viral proteins and the viral genome is replicated. C The viral genome and viral proteins assemble the nucleoprotein in preparation for ( D ) release of progeny into the surroundings with concomitant viral incorporation of host cell membrane. E Cellular and viral proteins expressed during ZIKV-LAV infection are also responsible for cell death in human GBM. F – G cleavage of caspase-3 leads to non-lytic or non-inflammatory cell death by apoptosis; H – I cleavage of gasdermin-D (GSDMD) leads to the formation of membrane pore complexes that shuttles inflammatory IL-1β outside of the cells. GSDMD cleavage also leads to inflammasome activation and lytic and inflammatory cell death by pyroptosis. Image created with BioRender.com

Journal: Journal of Translational Medicine

Article Title: Repurposing of Zika virus live-attenuated vaccine (ZIKV-LAV) strains as oncolytic viruses targeting human glioblastoma multiforme cells

doi: 10.1186/s12967-024-04930-4

Figure Lengend Snippet: Proposed model of human GBM cell death mediated by ZIKV-LAV infection. A – D General virus infection life cycle. A ZIKV-LAV enters human GBM cells through Axl and integrin α v β 5 cellular receptors. B The ZIKV-LAV genome is translated to express viral proteins and the viral genome is replicated. C The viral genome and viral proteins assemble the nucleoprotein in preparation for ( D ) release of progeny into the surroundings with concomitant viral incorporation of host cell membrane. E Cellular and viral proteins expressed during ZIKV-LAV infection are also responsible for cell death in human GBM. F – G cleavage of caspase-3 leads to non-lytic or non-inflammatory cell death by apoptosis; H – I cleavage of gasdermin-D (GSDMD) leads to the formation of membrane pore complexes that shuttles inflammatory IL-1β outside of the cells. GSDMD cleavage also leads to inflammasome activation and lytic and inflammatory cell death by pyroptosis. Image created with BioRender.com

Techniques: Infection, Virus, Membrane, Activation Assay

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