integrin αvβ5  (R&D Systems)

Journal: Cells

Article Title: WISP-1 Regulates Cardiac Fibrosis by Promoting Cardiac Fibroblasts’ Activation and Collagen Processing

doi: 10.3390/cells13110989

Figure Lengend Snippet: WISP-1 protein promoted Akt phosphorylation via integrin β1/FAK/ILK in human cardiac fibroblasts (HCFs). HCFs were cultured in supplemented fibroblast growth medium for 24 h and then starved in serum-free medium (SFM) for 48 h. The medium was replaced with fresh SFM in the presence or absence of recombinant human WISP-1 protein (500 ng/mL) for 30 min before cell lysis. Cell lysate samples were analysed by Western blotting using phosphorylated Akt (p-Akt) (Ser473) and total Akt (t-Akt) antibodies. ( A ) Representative Western blots of p-Akt (Ser473) and t-Akt protein expression. The ratio of p-Akt (Ser473) to t-Akt was calculated and expressed as the relative fold change to the control. Data shown as mean ± SEM (n = 9). Statistical analysis was performed using Mann–Whitney U test. * indicates p < 0.05. ( B ) Representative Western blots of p-Akt (Ser473) and t-Akt protein expression. The ratio of p-Akt (Ser473) to t-Akt was calculated and expressed as the relative fold change to WISP-1 + mouse non-immune IgG 1 control (mIgG) group. HCFs were pre-incubated with integrin β1-blocking antibodies (mouse IgG 1 clone) (β1 mAb, 10 μg/mL), integrin αVβ5-blocking antibodies (mouse IgG 1 clone) (αVβ5 mAb, 10 μg/mL), and mIgG control antibodies (10 μg/mL), respectively, for 30 min prior to WISP-1 protein treatment. Data shown as mean ± SEM (n = 5). Statistical analysis was performed using Kruskal–Wallis H test. * indicates p < 0.05. ( C ) Representative Western blots of p-Akt (Ser473) and t-Akt protein expression. The ratio of p-Akt (Ser473) to t-Akt was calculated and expressed as the relative fold change to WISP-1 group. HCFs were pre-incubated with defactinib (5 μM) or CPD22 (2.5 μM) for 30 min prior to WISP-1 protein treatment. Data shown as mean ± SEM (n = 4). Statistical analysis was performed using Mann–Whitney U test. * indicates p < 0.05. Approximate molecular weights in kDa are indicated adjacent to representative immunoblots.

Article Snippet: In some experiments, HCFs were pre-incubated with a mouse IgG 1 clone antibody (mAb) (10 μg/mL integrin β1 mAb [Biotechne, MAB177781], 10 μg/mL integrin αVβ5 mAb [Biotechne, MAB2528], or 10 μg/mL mouse IgG 1 control antibody [Biotechne, MAB002]), or an inhibitor (5μM defactinib [Abcam, Cambridge, UK, ab254452], 2.5 μM CPD22 [Merck, Darmstadt, Germany, 407331], or 25 μM GM6001 [Tocris, Bristol, UK, 2983]) for 30 min prior to WISP-1 protein treatment.

Techniques: Cell Culture, Recombinant, Lysis, Western Blot, Expressing, MANN-WHITNEY, Incubation, Blocking Assay

Journal: Cells

Article Title: WISP-1 Regulates Cardiac Fibrosis by Promoting Cardiac Fibroblasts’ Activation and Collagen Processing

doi: 10.3390/cells13110989

Figure Lengend Snippet: A schematic summary of the findings of this study. WISP-1 promotes cardiac fibroblasts’ phenotypic switch from quiescent fibroblasts to myofibroblasts (activated fibroblasts), promoting collagen processing and accumulation. WISP-1 activates Akt signalling via integrin β1/FAK/ILK in cardiac fibroblasts. Deletion of WISP-1 attenuates angiotensin II (AngII)-induced cardiac fibrotic remodelling in vivo. Figure key is illustrated on the top left-hand side of the figure. Purple ↓ denotes promotion; black ↑ denotes increase; ┤ denotes inhibition.

Article Snippet: In some experiments, HCFs were pre-incubated with a mouse IgG 1 clone antibody (mAb) (10 μg/mL integrin β1 mAb [Biotechne, MAB177781], 10 μg/mL integrin αVβ5 mAb [Biotechne, MAB2528], or 10 μg/mL mouse IgG 1 control antibody [Biotechne, MAB002]), or an inhibitor (5μM defactinib [Abcam, Cambridge, UK, ab254452], 2.5 μM CPD22 [Merck, Darmstadt, Germany, 407331], or 25 μM GM6001 [Tocris, Bristol, UK, 2983]) for 30 min prior to WISP-1 protein treatment.

Techniques: In Vivo, Inhibition

Journal: Journal of extracellular vesicles

Article Title: MFGE-8, a Corona Protein on Extracellular Vesicles, Mediates Self-Renewal and Survival of Human Pluripotent Stem Cells.

doi: 10.1002/jev2.70056

Figure Lengend Snippet: FIGURE 5 MFGE-8 facilitates EV uptake into recipient hESCs through integrin αvβ5. (a,b) The uptake of PKH-labelled EVs (30 µg/mL) that were pre-incubated with different concentrations of rhMFGE-8 (0–50 µg/mL) (a, n = 3) or anti-M8nAb (b, n = 4) for 1 h and then exposed to hESCs. The mean fluorescence intensity of hESC colonies was measured after 6 h. Cells were stained for OCT4 and E-cadherin, and nuclei were stained with DAPI. (c) Changes in the uptake of PKH-EVs (30 µg/mL) that were pre-incubated with or without rhMFGE-8 (25 µg/mL) for 1 h and exposed to hESCs for 6 h. anti- M8nAb (5 µg/mL) was added to the hESC culture for 1 h before the cells were exposed to PKH-EVs (n = 4). Cells were stained for OCT4 and E-cadherin, and nuclei were stained with DAPI. d, hESCs were exposed to untreated PKH-labelled bare EVs (30 µg/mL) or pre-treated for 3 h with hESC-derived SFs (bare EVs + SFs) or with hESC-derived SFs in which MFGE-8 activity was inhibited by anti-M8nAb (50 µg/mL). The mean fluorescence intensity was measured after 6 h of exposure (n = 4). Cells were stained for OCT4, and nuclei were stained with DAPI. (e) hESCs were exposed to PKH-EVs (30

Article Snippet: The following primary antibodies were used: mouse anti-OCT4 (1:400; sc-5279, Santa Cruz Biotechnology), rabbit anti-vimentin (1:200; 5741, Cell Signalling Technology), goat anti-E-cadherin (1:400; AF648, R&D Systems), mouse anti-integrin αvβ5 (1:100; MAB2528, R&D Systems), mouse anti-8-oxoguanine (1:200; MAB3560, Millipore) and rabbit anti-53BP1 (1:200; 4937, Cell Signalling Technology).

Techniques: Incubation, Fluorescence, Staining, Derivative Assay, Activity Assay

Journal: Journal of extracellular vesicles

Article Title: MFGE-8, a Corona Protein on Extracellular Vesicles, Mediates Self-Renewal and Survival of Human Pluripotent Stem Cells.

doi: 10.1002/jev2.70056

Figure Lengend Snippet: FIGURE 6 MFGE-8 mediates EV uptake in hESCs through the integrin αvβ5/Akt/GSK3β pathway. (a) A human phospho-kinase antibody array for hESCs cultured for 2 h in the absence or presence of anti-M8nAb (5 µg/mL) to block EV uptake. (b,c) Immunoblot showing phosphorylation of AKT and GSK3β after hESCs were treated with rhMFGE-8 (b) or hESC-derived EVs (c), with relative ratios of phosphorylation of AKT and GSK3β after 1 h of treatment shown on the right (n = 3). (d–f) Immunoblots showing the phosphorylation of AKT and GSK3β after hESCs were treated with different combinations of rhMFGE-8 (5 µg/mL), EVs (50 µg/mL), anti-M8nAb (5 µg/mL), anti-integrin αvβ5 antibody (anti-ITG αvβ5, 10 µg/mL), LY294002 (10 µM) and/or CHIR99021 (2 µM). hESCs were pre-incubated with or without anti-M8nAb, anti-integrin αvβ5 Ab, LY294002 and CHIR99021 for 1 h. Cells were treated with rhMFGE-8 or hESC-EVs for 15 min and 30 min, respectively. Relative phosphorylation is shown as a bar graph on the right (n = 3). (g) Image of PKH-EV uptake in hESCs treated with the factors indicated. PKH-labelled EVs (30 µg/mL) were pre-incubated with or without anti-M8nAb (25 µg/mL) for 1 h and exposed to hESCs for 6 h. The anti-ITG αvβ5 (10 µg/mL) or LY294002 (10 µM) was added to the hESC culture for 1 h before the cells were exposed to the PKH-EVs. Cells were stained for OCT4 and E-cadherin, and nuclei were stained with DAPI. Relative EV uptake is shown as a

Article Snippet: The following primary antibodies were used: mouse anti-OCT4 (1:400; sc-5279, Santa Cruz Biotechnology), rabbit anti-vimentin (1:200; 5741, Cell Signalling Technology), goat anti-E-cadherin (1:400; AF648, R&D Systems), mouse anti-integrin αvβ5 (1:100; MAB2528, R&D Systems), mouse anti-8-oxoguanine (1:200; MAB3560, Millipore) and rabbit anti-53BP1 (1:200; 4937, Cell Signalling Technology).

Techniques: Ab Array, Cell Culture, Blocking Assay, Western Blot, Phospho-proteomics, Derivative Assay, Incubation, Staining

Journal: Journal of extracellular vesicles

Article Title: MFGE-8, a Corona Protein on Extracellular Vesicles, Mediates Self-Renewal and Survival of Human Pluripotent Stem Cells.

doi: 10.1002/jev2.70056

Figure Lengend Snippet: FIGURE 7 MFGE-8 promotes endocytosis of EVs and self-renewal of hESCs by activating dynamin-1 and cyclin D1 via the integrin αvβ5/Akt/GSK3β axis. (a) Relative EV uptake in hESC cells treated with endocytosis inhibitors for 1 h (n = 3). hESC cells were incubated with PKH-EVs (30 µg/mL) for 6 h. (b) Images and quantitation of PKH-EV uptake in hESCs. EVs were pre-incubated with or without rhMFGE-8 (25 µg/mL) for 1 h before they were used to treat hESCs in the presence or absence of dynasore (DNS, 10 µM) (n = 4). Cells were stained for OCT4 and E-cadherin, and nuclei were stained with DAPI. (c) RT-qPCR analysis of DNM1 and DNM2 expression in hESCs (n = 3). (d) EV uptake in hESCs after siRNA knockdown of dynamin 1 (siDNM1) and dynamin 2 (siDNM2). Cell nuclei were stained with DAPI. Two siRNAs (#1 and #2) targeting different mRNA sequences of dynamins were used (n = 4). (e) Immunoblots showing phosphorylation of DNM1 in hESCs treated with or without 100 µg/mL hESC-EVs for 1 h. The relative phosphorylation of DNM1, quantitated by densitometry, is shown as a bar graph on the left (n = 3). (f) Images and quantitation of EV uptake for hESCs treated with combinations of anti-M8nAb and inhibitors, as indicated. hESCs were pre-treated with LY294002 (10 µM), CHIR99021 (2 µM), DNS (10 µM) or chlorpromazine (CPZ) (1 µM). hESC-EVs were pre-incubated with or without anti-M8nAb (25 µg/mL) for 1 h before exposure to cells (n = 3). Cells

Article Snippet: The following primary antibodies were used: mouse anti-OCT4 (1:400; sc-5279, Santa Cruz Biotechnology), rabbit anti-vimentin (1:200; 5741, Cell Signalling Technology), goat anti-E-cadherin (1:400; AF648, R&D Systems), mouse anti-integrin αvβ5 (1:100; MAB2528, R&D Systems), mouse anti-8-oxoguanine (1:200; MAB3560, Millipore) and rabbit anti-53BP1 (1:200; 4937, Cell Signalling Technology).

Techniques: Incubation, Quantitation Assay, Staining, Quantitative RT-PCR, Expressing, Knockdown, Western Blot, Phospho-proteomics

Journal: bioRxiv

Article Title: Overcoming Immune Checkpoint Inhibitor Resistance via Potent and Selective Dual αvβ6/8 Inhibitors Based on Engineered Lasso Peptides

doi: 10.1101/2025.01.28.635346

Figure Lengend Snippet:

Article Snippet: Human integrins αvβ1, αvβ3, αvβ5, αvβ6, αvβ8, α5β1, and αIIbβ3 were purchased from R&D Systems (Minneapolis, MN) or Acro Biosystems (Newark, DE, USA).

Techniques: Inhibition, Enzyme-linked Immunosorbent Assay, Positive Control

Journal: bioRxiv

Article Title: ANGPTL6 variant induces cerebral vascular dysfunction and predisposes to intracranial aneurysm in mice

doi: 10.1101/2025.01.10.632391

Figure Lengend Snippet: A. Representative curve of cell index measurement for rat VSMC adhering on gelatin coating without (grey) and with ANGPTL6 (4 ng/mL, blue). B . Quantification of the rate of VSMC adhesion on gelatin coating without or with ANGPTL6 (1-4 ng/mL) using the slope of the cell index curve in its linear part (One-way ANOVA; *** P < 0.001). C. Representative images of the evan blue staining of rat VSMC that transmigrated across a porous membrane coated with gelatin containing or not ANGPTL6 after 24 h (left) and the corresponding quantification of the area covered by rat VSMC (normalized to the gelatin alone condition, Wilcoxon t -test) D. Representative curve of cell index measurement for human VSMC adhering on gelatin coating without (grey) and with ANGPTL6 (4 ng/mL, blue) (left) and corresponding quantification of the rate of adhesion using the slope of the cell index curve in its linear part (Wilcoxon t -test; * P <0.05). F. Single-cycle kinetic SPR analysis of the ANGPTL6 interaction with αVβ5 or α5β1 integrins (12.5 nM; 25 nM; 50 nM; 100nM and 200 nM) or fibronectin (62.5 nM; 125 nM; 250 nM; 500 nM and 1000 nM). The red line represents the kinetic global fit of the ANGPTL6-αVβ5 interaction using 1:1 Langmuir model.

Article Snippet: SPR was used to assess the interaction between fibronectin (ECM001, Sigma-aldrich), αVβ5 integrin (2528-AV, R&D systems) and α5β1 integrin (3230-A5, R&D systems) with ANGPTL6 (CSB-MP822824HU, Cusabio) immobilized onto CM5 (1000 RU; GE Healthcare) using a Biacore T200 (GE Healthcare).

Techniques: Staining, Membrane