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anti human αvβ5 integrin antibody (R&D Systems)

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R&D Systems anti human αvβ5 integrin antibody
Anti Human αvβ5 Integrin Antibody, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/anti+human+%CE%B1v%CE%B25+integrin+antibody/Human+Integrin+alpha+V+beta+5+APC-conjugated+Antibody/pm41319961-60-7-25
Average 93 stars, based on 10 article reviews

anti human αvβ5 integrin antibody - by Bioz Stars, 2026-09

93/100 stars

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Article Title: Tumor-targeted adeno associated virus-loaded complexes comprising tannic acid and phenylboronic acid-polymers for orthotopic glioblastoma therapy.
Article Snippet: Dulbecco’s Modified Eagle Medium (DMEM), Roswell Park Memorial Institute Medium (RPMI), penicillin-streptomycin (PS), Trypsin-EDTA solution and deuterium oxide (D2O) were purchased from Sigma-Aldrich (Merck, St. Louis, MO, USA). .. FITC-conjugated anti-human αvβ3 integrin antibody (FAB3050G), APC-conjugated anti-human αvβ5 integrin antibody (FAB2528A), Alexa Fluor-conjugated isotype control IgG, and purified human IgG (1–001-A) were purchased from R&D Systems (Minneapolis, MN, USA). .. Accutase (AT104) was obtained from Funakoshi Co., Ltd. (Tokyo, Japan).

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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.

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

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.

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.

Techniques: In Vivo, Inhibition

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

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

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

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

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

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

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

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

Evaluation of αvβ6-dependent activation of chimeric G115 γδ TCRs. ( A ) Analysis of integrin expression on A375 puro and A375-β6 cells. Red–integrin; blue–isotype control. Data are representative of 3 independent replicates. Co-cultures were performed between A375 puro ( B ) and A375-β6 ( C ) tumour cells and untransduced (untrans) or transduced T-cell populations at an effector-to-target ratio of 1:1 for 72 h. Residual tumour cell viability was determined using an MTT assay (mean ± SEM of indicated replicates). Statistical analysis was performed using one-way ANOVA; **** p < 0.0001. ( D ) Supernatants collected from co-cultures described in ( B , C ) were analysed for IFN-γ by ELISA (mean ± SEM; n = 21–45 from 4–6 independent donors). Statistical analysis was performed using one-way ANOVA; * p < 0.05; ** p < 0.01.

Journal: Biology

Article Title: Engineering a Dual Specificity γδ T-Cell Receptor for Cancer Immunotherapy

doi: 10.3390/biology13030196

Figure Lengend Snippet: Evaluation of αvβ6-dependent activation of chimeric G115 γδ TCRs. ( A ) Analysis of integrin expression on A375 puro and A375-β6 cells. Red–integrin; blue–isotype control. Data are representative of 3 independent replicates. Co-cultures were performed between A375 puro ( B ) and A375-β6 ( C ) tumour cells and untransduced (untrans) or transduced T-cell populations at an effector-to-target ratio of 1:1 for 72 h. Residual tumour cell viability was determined using an MTT assay (mean ± SEM of indicated replicates). Statistical analysis was performed using one-way ANOVA; **** p < 0.0001. ( D ) Supernatants collected from co-cultures described in ( B , C ) were analysed for IFN-γ by ELISA (mean ± SEM; n = 21–45 from 4–6 independent donors). Statistical analysis was performed using one-way ANOVA; * p < 0.05; ** p < 0.01.

Article Snippet: Integrin expression was detected by flow cytometry using the following antibodies: αvβ3 integrin (FAB3050A, R&D Systems, Minneapolis, MN, USA), αvβ5 integrin (FAB2528A, R&D systems), αvβ6 integrin (FAB4155A, R&D Systems) and αvβ8 integrin (FAB4775A, R&D systems).

Techniques: Activation Assay, Expressing, Control, MTT Assay, Enzyme-linked Immunosorbent Assay

Evaluation of PAg-dependent activation of chimeric G115 γδ TCRs by ffLuc-expressing K562 cells. ( A ) Analysis of αvβ6 integrin expression on ffLuc + K562 cells. Red–integrin; blue–isotype control. Data are representative of 3 independent replicates. ( B ) Firefly luciferase-expressing K562 cells were pre-incubated with the indicated Zol concentration for 24 h prior to the establishment of co-cultures with untrans(duced) or transduced T-cell populations at an effector to target ratio 1:1 for 72 h. Data show mean ± SEM of residual K562 viability (n = 6–11 from 4 independent donors), as determined by luciferase assay. Statistical analysis was performed using two-way ANOVA; *** p < 0.001; **** p < 0.0001. ( C ) Supernatants collected from co-cultures described in B were analysed for IFN-γ by ELISA (mean ± SEM; n = 19 from 3 independent donors). Statistical analysis was performed using two-way ANOVA; *** p < 0.001; **** p < 0.0001.

Journal: Biology

Article Title: Engineering a Dual Specificity γδ T-Cell Receptor for Cancer Immunotherapy

doi: 10.3390/biology13030196

Figure Lengend Snippet: Evaluation of PAg-dependent activation of chimeric G115 γδ TCRs by ffLuc-expressing K562 cells. ( A ) Analysis of αvβ6 integrin expression on ffLuc + K562 cells. Red–integrin; blue–isotype control. Data are representative of 3 independent replicates. ( B ) Firefly luciferase-expressing K562 cells were pre-incubated with the indicated Zol concentration for 24 h prior to the establishment of co-cultures with untrans(duced) or transduced T-cell populations at an effector to target ratio 1:1 for 72 h. Data show mean ± SEM of residual K562 viability (n = 6–11 from 4 independent donors), as determined by luciferase assay. Statistical analysis was performed using two-way ANOVA; *** p < 0.001; **** p < 0.0001. ( C ) Supernatants collected from co-cultures described in B were analysed for IFN-γ by ELISA (mean ± SEM; n = 19 from 3 independent donors). Statistical analysis was performed using two-way ANOVA; *** p < 0.001; **** p < 0.0001.

Techniques: Activation Assay, Expressing, Control, Luciferase, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay

Evaluation of the anti-tumour activity of chimeric G115 γδ TCRs against BxPC3 pancreatic tumour cells. ( A ) Analysis of integrin expression on BxPC3 cells. Red–integrin; blue–isotype control. Data are representative of 3 independent replicates. Co-cultures were performed between BxPC3 tumour cells (No Zol; B ) or Zol-sensitised BxPC3 tumour cells (+Zol; C ) and untrans(duced) or transduced T-cell populations at an effector to target ratio of 1:1 for 72 h. Tumour cell viability was determined using an MTT assay (mean ± SEM of indicated replicates). Statistical analysis was performed using one-way ANOVA; **** p < 0.0001, * p < 0.05, N/S–not significant.

Journal: Biology

Article Title: Engineering a Dual Specificity γδ T-Cell Receptor for Cancer Immunotherapy

doi: 10.3390/biology13030196

Figure Lengend Snippet: Evaluation of the anti-tumour activity of chimeric G115 γδ TCRs against BxPC3 pancreatic tumour cells. ( A ) Analysis of integrin expression on BxPC3 cells. Red–integrin; blue–isotype control. Data are representative of 3 independent replicates. Co-cultures were performed between BxPC3 tumour cells (No Zol; B ) or Zol-sensitised BxPC3 tumour cells (+Zol; C ) and untrans(duced) or transduced T-cell populations at an effector to target ratio of 1:1 for 72 h. Tumour cell viability was determined using an MTT assay (mean ± SEM of indicated replicates). Statistical analysis was performed using one-way ANOVA; **** p < 0.0001, * p < 0.05, N/S–not significant.

Techniques: Activity Assay, Expressing, Control, MTT Assay

Evaluation of the anti-tumour activity of chimeric G115 γδ TCRs against Panc1 pancreatic tumour cells. ( A ) Analysis of integrin expression on Panc1 cells. Red–integrin; blue–isotype control. Data are representative of 3 independent replicates. Co-cultures were performed between Panc1 tumour cells (No Zol; B ) or Zol-sensitised Panc1 tumour cells (+ Zol; C ) and untransduced (untrans) or transduced T-cell populations at an effector to target ratio of 1:1 for 72 h. Tumour cell viability was determined using an MTT assay (mean ± SEM of indicated replicates). Statistical analysis was performed using one-way ANOVA; **** p < 0.0001, ** p < 0.01, N/S–not significant.

Journal: Biology

Article Title: Engineering a Dual Specificity γδ T-Cell Receptor for Cancer Immunotherapy

doi: 10.3390/biology13030196

Figure Lengend Snippet: Evaluation of the anti-tumour activity of chimeric G115 γδ TCRs against Panc1 pancreatic tumour cells. ( A ) Analysis of integrin expression on Panc1 cells. Red–integrin; blue–isotype control. Data are representative of 3 independent replicates. Co-cultures were performed between Panc1 tumour cells (No Zol; B ) or Zol-sensitised Panc1 tumour cells (+ Zol; C ) and untransduced (untrans) or transduced T-cell populations at an effector to target ratio of 1:1 for 72 h. Tumour cell viability was determined using an MTT assay (mean ± SEM of indicated replicates). Statistical analysis was performed using one-way ANOVA; **** p < 0.0001, ** p < 0.01, N/S–not significant.

Techniques: Activity Assay, Expressing, Control, MTT Assay

αvβ5-integrin and chondroitin sulfate proteoglycans are implicated in Wnt1-inducible signaling protein 1 (WISP1)-induced production of migration inhibitory factor (MIF) from human lung fibroblasts (HLFs). HLFs were pretreated for 30 minutes with anti-αvβ5-integrin antibody (6 µg/mL) ( A ) or for 2 h with chondroitinase ABC or chondroitinase AC II (3 units/mL) ( B ), followed by stimulation with WISP1 (100 ng/mL) for 48 h, and the levels of MIF in conditioned media were determined by ELISA. Data represent the means ± SD ( n = 3). *Statistically significant increase ( P < 0.05) compared to control. #Statistically significant decrease ( P < 0.05) compared to WISP1-treated cells. IgG represents immunoglobulins from nonimmunized mouse.

Journal: American Journal of Physiology - Cell Physiology

Article Title: WISP1 induces the expression of macrophage migration inhibitory factor in human lung fibroblasts through Src kinases and EGFR-activated signaling pathways

doi: 10.1152/ajpcell.00410.2023

Figure Lengend Snippet: αvβ5-integrin and chondroitin sulfate proteoglycans are implicated in Wnt1-inducible signaling protein 1 (WISP1)-induced production of migration inhibitory factor (MIF) from human lung fibroblasts (HLFs). HLFs were pretreated for 30 minutes with anti-αvβ5-integrin antibody (6 µg/mL) ( A ) or for 2 h with chondroitinase ABC or chondroitinase AC II (3 units/mL) ( B ), followed by stimulation with WISP1 (100 ng/mL) for 48 h, and the levels of MIF in conditioned media were determined by ELISA. Data represent the means ± SD ( n = 3). *Statistically significant increase ( P < 0.05) compared to control. #Statistically significant decrease ( P < 0.05) compared to WISP1-treated cells. IgG represents immunoglobulins from nonimmunized mouse.

Article Snippet: Recombinant human EGF and anti-integrin αvβ5 mouse mAb (no. MAB2528-SP) were purchased from R&D Systems (Bio-Techne Ltd., Abingdon, UK).

Techniques: Migration, Enzyme-linked Immunosorbent Assay, Control

Proposed model of the effects of Wnt1-inducible signaling protein 1 (WISP1) on the expression of migration inhibitory factor (MIF) and its receptors, as well as on the expression of cyclooxygenase (COX)-2, IL-6, and matrix metalloproteinase (MMP)-2, and production of prostaglandin E 2 (PGE 2 ) from human lung fibroblasts (HLFs). The effect of WISP-1 on the expression of MIF in HLFs with the complex interactions between WISP-1, αvβ5, Src kinases, EGF receptor (EGFR), and CD74/CD44 receptors, reveal intricate regulatory networks that could contribute to the expression of proinflamamtory molecules and remodeling enzymes secretion.

Journal: American Journal of Physiology - Cell Physiology

Article Title: WISP1 induces the expression of macrophage migration inhibitory factor in human lung fibroblasts through Src kinases and EGFR-activated signaling pathways

doi: 10.1152/ajpcell.00410.2023

Figure Lengend Snippet: Proposed model of the effects of Wnt1-inducible signaling protein 1 (WISP1) on the expression of migration inhibitory factor (MIF) and its receptors, as well as on the expression of cyclooxygenase (COX)-2, IL-6, and matrix metalloproteinase (MMP)-2, and production of prostaglandin E 2 (PGE 2 ) from human lung fibroblasts (HLFs). The effect of WISP-1 on the expression of MIF in HLFs with the complex interactions between WISP-1, αvβ5, Src kinases, EGF receptor (EGFR), and CD74/CD44 receptors, reveal intricate regulatory networks that could contribute to the expression of proinflamamtory molecules and remodeling enzymes secretion.

Article Snippet: Recombinant human EGF and anti-integrin αvβ5 mouse mAb (no. MAB2528-SP) were purchased from R&D Systems (Bio-Techne Ltd., Abingdon, UK).

Techniques: Expressing, Migration

( a ) RFP growth competition assay (used in Figs. , , ): The ipUSEPR vector expresses a sgRNA together with a puromycin-resistant gene (PuroR) and a TagRFP fluorescent protein. The RFP fluorescent signal of live (DAPI – ) singlet cells was detected by an Attune NxT flow cytometer with an HTS autosampler. The sgRNA targeting a functionally important gene will result in a reduced RFP + population in the culture. ( b ) Gating strategy for detecting the cell surface αVβ5 expression (used in Fig. ).

Journal: Nature Structural & Molecular Biology

Article Title: A novel class of inhibitors that disrupts the stability of integrin heterodimers identified by CRISPR-tiling-instructed genetic screens

doi: 10.1038/s41594-024-01211-y

Figure Lengend Snippet: ( a ) RFP growth competition assay (used in Figs. , , ): The ipUSEPR vector expresses a sgRNA together with a puromycin-resistant gene (PuroR) and a TagRFP fluorescent protein. The RFP fluorescent signal of live (DAPI – ) singlet cells was detected by an Attune NxT flow cytometer with an HTS autosampler. The sgRNA targeting a functionally important gene will result in a reduced RFP + population in the culture. ( b ) Gating strategy for detecting the cell surface αVβ5 expression (used in Fig. ).

Article Snippet: Cell surface integrin αVβ5 was recognized by a mouse monoclonal anti-human αVβ5 antibody (clone P1F76; sc-13588, Santa Cruz Biotech; 1:200) and stained with AF488-conjugated donkey anti-mouse IgG (ab150105, Abcam) secondary antibody.

Techniques: Competitive Binding Assay, Plasmid Preparation, Flow Cytometry, Expressing

a , Model of integrin α (red) and β (blue) subunits and domain structures. The binding site of the extracellular ligand (yellow) is assembled upon heterodimerization of the α/β subunits. b , Schematic outline of integrin family CRISPR screens (712 sgRNAs) in Cas9-expressing MDA231 and PANC1 cells. c , Fold change of each sgRNA from day 0 to day 24 in MDA231-Cas9 + ( x axis) and PANC1-Cas9 + ( y axis) cells. The sgRNAs targeting ITGAV (red dots), ITGB5 (blue dots), positive controls (yellow triangles), negative controls (green triangles) and the total library (gray dots) are indicated. d , Heatmap showing CRISPR impact scores (median log 10 fold change of 25 sgRNAs) of each integrin subunit in the integrin network consisting of 24 distinct integrin α/β heterodimers. The solid lines indicate the integrin α/β pairs forming the RGD receptors (yellow), collagen receptors (pink), laminin receptors (brown) and leukocyte-specific receptors (green). The red dotted circle highlights αVβ5 as the top essential integrin heterodimer in cancer cells. e , Growth competition assay of MDA231-Cas9 + cells transduced with RFP-labeled sgCtrl (gray lines; two independent sgRNA sequences) and sgITGB1/3/5/6/8 (blue lines; three independent sgRNA sequences for each gene). Asterisk indicates that all three sgRNAs for each ITGB gene group were significantly different ( P < 0.01) from the two sgCtrl groups ( n = 3 for each group). f , Western blot of ITGB5 and β-actin in MDA231-Cas9 + cells transduced with sgCtrl ( n = 2 independent sgRNA sequences) and sgITGB5 ( n = 3 independent sgRNA sequences) for 3 days. g , h , Cellular apoptosis detected by Annexin V + /DAPI − ( g ) and cell cycle monitored by EdU incorporation ( h ) in MDA231-Cas9 + cells transduced with sgCtrl and sgITGB5 for 3 days ( n = 3 for each group). i , Gene ranking based on the Pearson coefficient ( r ) of CERES scores between ITGAV and ITGB5 (blue) compared with other ITGAV partner β subunit genes ITGB1 / 3 / 6 / 8 (yellow) in the 769 tested cell models (Extended Data Fig. ). Data are presented as the mean ± s.e.m. P values were calculated by two-sided Student’s t -test.

Journal: Nature Structural & Molecular Biology

Article Title: A novel class of inhibitors that disrupts the stability of integrin heterodimers identified by CRISPR-tiling-instructed genetic screens

doi: 10.1038/s41594-024-01211-y

Figure Lengend Snippet: a , Model of integrin α (red) and β (blue) subunits and domain structures. The binding site of the extracellular ligand (yellow) is assembled upon heterodimerization of the α/β subunits. b , Schematic outline of integrin family CRISPR screens (712 sgRNAs) in Cas9-expressing MDA231 and PANC1 cells. c , Fold change of each sgRNA from day 0 to day 24 in MDA231-Cas9 + ( x axis) and PANC1-Cas9 + ( y axis) cells. The sgRNAs targeting ITGAV (red dots), ITGB5 (blue dots), positive controls (yellow triangles), negative controls (green triangles) and the total library (gray dots) are indicated. d , Heatmap showing CRISPR impact scores (median log 10 fold change of 25 sgRNAs) of each integrin subunit in the integrin network consisting of 24 distinct integrin α/β heterodimers. The solid lines indicate the integrin α/β pairs forming the RGD receptors (yellow), collagen receptors (pink), laminin receptors (brown) and leukocyte-specific receptors (green). The red dotted circle highlights αVβ5 as the top essential integrin heterodimer in cancer cells. e , Growth competition assay of MDA231-Cas9 + cells transduced with RFP-labeled sgCtrl (gray lines; two independent sgRNA sequences) and sgITGB1/3/5/6/8 (blue lines; three independent sgRNA sequences for each gene). Asterisk indicates that all three sgRNAs for each ITGB gene group were significantly different ( P < 0.01) from the two sgCtrl groups ( n = 3 for each group). f , Western blot of ITGB5 and β-actin in MDA231-Cas9 + cells transduced with sgCtrl ( n = 2 independent sgRNA sequences) and sgITGB5 ( n = 3 independent sgRNA sequences) for 3 days. g , h , Cellular apoptosis detected by Annexin V + /DAPI − ( g ) and cell cycle monitored by EdU incorporation ( h ) in MDA231-Cas9 + cells transduced with sgCtrl and sgITGB5 for 3 days ( n = 3 for each group). i , Gene ranking based on the Pearson coefficient ( r ) of CERES scores between ITGAV and ITGB5 (blue) compared with other ITGAV partner β subunit genes ITGB1 / 3 / 6 / 8 (yellow) in the 769 tested cell models (Extended Data Fig. ). Data are presented as the mean ± s.e.m. P values were calculated by two-sided Student’s t -test.

Techniques: Binding Assay, CRISPR, Expressing, Competitive Binding Assay, Transduction, Labeling, Western Blot

a , 3D ‘docking box’ (cube) defined by the CRISPR-hypersensitive regions (numbers 1–7) within the ITGAV β-propeller domain. b , Compound (Cpd) ranking based on free binding energy (ΔG°) to the ‘docking box’ within the β-propeller domain predicted by AutoDock Vina. c , d , Heatmap showing relative CellTiter Glo (left) and CCK8 (right) signals (percentage of the signal for dimethyl sulfoxide; DMSO) in MDA231 cells incubated with 10 µM of 500 selected compounds ( c ) and the top nine effective compounds ( d ) for 3 days. Effective cell killing was defined as less than 10% relative signals for both CellTiter Glo and CCK8 assays. e , Schematic outline of flow cytometric measurement of cell surface integrin αVβ5 using a monoclonal antibody against integrin αVβ5 heterodimers. f , Effects of the top nine candidate compounds on cell surface integrin αVβ5 levels upon 1 h compound treatments ( n = 4 for each condition). g , h , Cellular apoptosis detected by Annexin V + /DAPI − ( g ) and cell cycle monitored by EdU incorporation ( h ) in MDA231 cells treated with Cpd_AV2 (40 µM) for 0 to 3 h ( n = 3 for each time point). i , Representative fluorescence images of F-actin (FITC, green) and nucleus (DAPI, blue) staining in MDA231 cells treated with control (DMSO) and Cpd_AV2 (40 µM) for 10 min. Scale bars, 20 µm. j , Violin plot showing the distribution of cell size (µm 2 ) in MDA231 cells treated with control (DMSO) and Cpd_AV2 (40 µM) for 10 min. Data are presented as the mean ± s.e.m. P values were calculated by two-sided Student’s t -test.

Journal: Nature Structural & Molecular Biology

Article Title: A novel class of inhibitors that disrupts the stability of integrin heterodimers identified by CRISPR-tiling-instructed genetic screens

doi: 10.1038/s41594-024-01211-y

Figure Lengend Snippet: a , 3D ‘docking box’ (cube) defined by the CRISPR-hypersensitive regions (numbers 1–7) within the ITGAV β-propeller domain. b , Compound (Cpd) ranking based on free binding energy (ΔG°) to the ‘docking box’ within the β-propeller domain predicted by AutoDock Vina. c , d , Heatmap showing relative CellTiter Glo (left) and CCK8 (right) signals (percentage of the signal for dimethyl sulfoxide; DMSO) in MDA231 cells incubated with 10 µM of 500 selected compounds ( c ) and the top nine effective compounds ( d ) for 3 days. Effective cell killing was defined as less than 10% relative signals for both CellTiter Glo and CCK8 assays. e , Schematic outline of flow cytometric measurement of cell surface integrin αVβ5 using a monoclonal antibody against integrin αVβ5 heterodimers. f , Effects of the top nine candidate compounds on cell surface integrin αVβ5 levels upon 1 h compound treatments ( n = 4 for each condition). g , h , Cellular apoptosis detected by Annexin V + /DAPI − ( g ) and cell cycle monitored by EdU incorporation ( h ) in MDA231 cells treated with Cpd_AV2 (40 µM) for 0 to 3 h ( n = 3 for each time point). i , Representative fluorescence images of F-actin (FITC, green) and nucleus (DAPI, blue) staining in MDA231 cells treated with control (DMSO) and Cpd_AV2 (40 µM) for 10 min. Scale bars, 20 µm. j , Violin plot showing the distribution of cell size (µm 2 ) in MDA231 cells treated with control (DMSO) and Cpd_AV2 (40 µM) for 10 min. Data are presented as the mean ± s.e.m. P values were calculated by two-sided Student’s t -test.

Techniques: CRISPR, Binding Assay, Incubation, Fluorescence, Staining, Control

a , Purification of bacterial-expressed recombinant ITGAV β-propeller domain (peptide region 31–492 aa; N-terminal His 6 -tagged) using immobilized metal affinity chromatography (IMAC) and anion exchange chromatography (IEX). The input and purified ITGAV β-propeller domain samples were visualized by gel electrophoresis and silver staining (right; gel representative of two independent protein purification experiments). b , Protein thermal stability as estimated by fluorescent dye incorporation of the purified ITGAV β-propeller domain under control (DMSO) and Cpd_AV2 (40 µM) conditions. c , Protein surface model (left) showing a docking simulation of the ITGAV β-propeller domain (colored by NCS) interacting with Cpd_AV2 (yellow). Protein ribbon model (right) illustrates an overlap of ITGB5 lysine 287 (within βA loop; cyan) and Cpd_AV2 (yellow) binding on the β-propeller HIP of ITGAV. d , e , Effects of cilengitide and Cpd_AV2 on cell surface integrin αVβ5 levels after 1 h treatment ( d ) and cell expansion after 72 h treatment in MDA231 cells ( e ) ( n = 3 for each group). f , Effects of 72 h Cpd_AV2 treatment on expansion of six cancer cell models ( n = 3 for each group). g , Chemical structure of Cpd_AV2 (source: NCI/DTP Open Chemicals Repository). h , Model showing distinct mechanisms of action between Cpd_AV2 (left) and cilengitide (right) for suppressing ECM-to-integrin αVβ5 signaling (middle). Data are presented as the mean ± s.e.m. P values were calculated by two-sided Student’s t -test. NSCLC, nonsmall-cell lung cancer.

Journal: Nature Structural & Molecular Biology

Article Title: A novel class of inhibitors that disrupts the stability of integrin heterodimers identified by CRISPR-tiling-instructed genetic screens

doi: 10.1038/s41594-024-01211-y

Figure Lengend Snippet: a , Purification of bacterial-expressed recombinant ITGAV β-propeller domain (peptide region 31–492 aa; N-terminal His 6 -tagged) using immobilized metal affinity chromatography (IMAC) and anion exchange chromatography (IEX). The input and purified ITGAV β-propeller domain samples were visualized by gel electrophoresis and silver staining (right; gel representative of two independent protein purification experiments). b , Protein thermal stability as estimated by fluorescent dye incorporation of the purified ITGAV β-propeller domain under control (DMSO) and Cpd_AV2 (40 µM) conditions. c , Protein surface model (left) showing a docking simulation of the ITGAV β-propeller domain (colored by NCS) interacting with Cpd_AV2 (yellow). Protein ribbon model (right) illustrates an overlap of ITGB5 lysine 287 (within βA loop; cyan) and Cpd_AV2 (yellow) binding on the β-propeller HIP of ITGAV. d , e , Effects of cilengitide and Cpd_AV2 on cell surface integrin αVβ5 levels after 1 h treatment ( d ) and cell expansion after 72 h treatment in MDA231 cells ( e ) ( n = 3 for each group). f , Effects of 72 h Cpd_AV2 treatment on expansion of six cancer cell models ( n = 3 for each group). g , Chemical structure of Cpd_AV2 (source: NCI/DTP Open Chemicals Repository). h , Model showing distinct mechanisms of action between Cpd_AV2 (left) and cilengitide (right) for suppressing ECM-to-integrin αVβ5 signaling (middle). Data are presented as the mean ± s.e.m. P values were calculated by two-sided Student’s t -test. NSCLC, nonsmall-cell lung cancer.

Techniques: Purification, Recombinant, Affinity Chromatography, Chromatography, Nucleic Acid Electrophoresis, Silver Staining, Protein Purification, Control, Binding Assay

( a ) 3D structure of the extracellular domain of ITGB5 was modeled by AlphaFold2 (cyan) and overlaid with the ITGB3 portion of integrin αVβ3 structure resolved by Xiong et al. (PDB ID: 3IJE, chain B; blue). Overall, we observed high concordance of the 3D structures between ITGB3 and ITGB5, including the highly conserved basic amino acid (ITGB3’s R287 or ITGB5’s K287) in the loop motif of the βA domain highlighted in ( b ). ( c ) Modeling of ITGAV/ITGB5 interaction using the AlphaFold2 predicted ITGB5 structure (cyan) and the ITGAV portion of integrin αVβ3 structure (PDB ID: 3IJE, chain A; red). ( d and e ) Molecular dynamics simulation using GROMACS 2022 with CHARMM36m force field indicates ( d ) a close contact between ITGB5’s K287 and ITGAV’s β-propeller HIP pocket (purple box), and ( e ) the occupancy of Cpd_AV2 (yellow) into ITGAV’s HIP pocket disengaged the side chain of ITGB5’s K287 from stably interacting with ITGAV. ( f ) Substitution of ITGB5’s K287 with an alanine (K287A) significantly attenuated the ITGAV/ITGB5 NanoBRET signal, highlighting an essential role of this basic residue in integrin αVβ5 assembly (n = 3 for each group). Data are represented as mean ± s.e.m. P value was calculated by two-sided Student’s t-test.

Journal: Nature Structural & Molecular Biology

Article Title: A novel class of inhibitors that disrupts the stability of integrin heterodimers identified by CRISPR-tiling-instructed genetic screens

doi: 10.1038/s41594-024-01211-y

Figure Lengend Snippet: ( a ) 3D structure of the extracellular domain of ITGB5 was modeled by AlphaFold2 (cyan) and overlaid with the ITGB3 portion of integrin αVβ3 structure resolved by Xiong et al. (PDB ID: 3IJE, chain B; blue). Overall, we observed high concordance of the 3D structures between ITGB3 and ITGB5, including the highly conserved basic amino acid (ITGB3’s R287 or ITGB5’s K287) in the loop motif of the βA domain highlighted in ( b ). ( c ) Modeling of ITGAV/ITGB5 interaction using the AlphaFold2 predicted ITGB5 structure (cyan) and the ITGAV portion of integrin αVβ3 structure (PDB ID: 3IJE, chain A; red). ( d and e ) Molecular dynamics simulation using GROMACS 2022 with CHARMM36m force field indicates ( d ) a close contact between ITGB5’s K287 and ITGAV’s β-propeller HIP pocket (purple box), and ( e ) the occupancy of Cpd_AV2 (yellow) into ITGAV’s HIP pocket disengaged the side chain of ITGB5’s K287 from stably interacting with ITGAV. ( f ) Substitution of ITGB5’s K287 with an alanine (K287A) significantly attenuated the ITGAV/ITGB5 NanoBRET signal, highlighting an essential role of this basic residue in integrin αVβ5 assembly (n = 3 for each group). Data are represented as mean ± s.e.m. P value was calculated by two-sided Student’s t-test.

Techniques: Stable Transfection, Residue

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