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

Journal: ACS Omega

Article Title: Design and Evaluation of Short Self-Assembling Depsipeptides as Bioactive and Biodegradable Hydrogels

doi: 10.1021/acsomega.7b01641

Figure Lengend Snippet: Half inhibition (i.e., IC 50 ) of the fluorescence of FITC-GRGDSP occurs at a ∼40-fold lower concentration for RGD than R-Glc-D, indicating greater affinity of RGD for integrin than R-Glc-D (A). Comparison of FP inhibition by R-Glc-D and RGE peptides (negative control). The average IC 50 value for R-Glc-D is 1115 ± 211 μM (mean ± standard deviation). RGE was tested once as a negative control, and the calculated IC 50 value was 2840 μM (B).

Article Snippet: Human recombinant integrin α 5 β 1 (R&D Systems) was diluted to 900 nM in the same Tris buffer.

Techniques: Inhibition, Fluorescence, Concentration Assay, Comparison, Negative Control, Standard Deviation

Journal: Journal of Cellular and Molecular Medicine

Article Title: Endostatin induces autophagy in endothelial cells by modulating Beclin 1 and β-catenin levels

doi: 10.1111/j.1582-4934.2009.00722.x

Figure Lengend Snippet: Effect of integrin antibodies and down-regulation of integrin sub-units on endostatin-induced autophagy. (A) LC3-GFP–transfected HUVECs were treated with P125A-endostatin (20 μg/ml) for 24 hrs in the presence of monoclonal antibody against integrin α 5 β 1 (15 μg/ml)( n = 3). LC3-GFP–transfected HUVECs treated with isotype matched, normal IgG was used as a control for the study. Notice that autophagy induction was partially inhibited by integrin α 5 β 1 antibody. (B) HUVECs were transfected with siRNA specific to integrin α 5 and integrin β 1 at a concentration of 40 nM. After 36 hrs of transfection, HUVECs were further transfected with LC3-GFP. Cells were subsequently treated with P125A-endostatin for 24 hrs and assessed for autophagic vesicles per cell ( n = 3). (C) Effect of 3-MA on P125A-endostatin induced autophagy in HUVECs. LC3-GFP–transfected HUVECs were treated with P125A-endostatin (20 μg/ml) for 24 hrs. 3-MA (5 mM) was added to LC3-GFP–transfected HUVEC culture 12 hrs into the experiment ( n = 3). (D) LC3-GFP was co-transfected with shRNA specific to Beclin 1 or control scrambled shRNA to HUVECs [ , 43]. Results are shown as mean ± S.E. Statistical significance was determined using Student’s t-test.

Article Snippet: Human integrin α 5 β 1 mAb was obtained from Chemicon (Temecula, CA).

Techniques: Transfection, Concentration Assay, shRNA

Journal: Tissue Engineering. Part A

Article Title: Linear Shear Conditioning Improves Vascular Graft Retention of Adipose-Derived Stem Cells by Upregulation of the ? 5 ? 1 Integrin

doi: 10.1089/ten.tea.2009.0238

Figure Lengend Snippet: Analysis of integrin α5β1 expression in ASCs under static and shear conditions. (A) Orbital shear stress increases ASC attachment to fibronectin-coated surfaces by 1.5-fold compared to static culture conditions. Attachment of static cultures is reduced by over 90% by α5β1 blockade. Shear cultures demonstrated an 80% reduction in attachment following blockade. (B) Integrin-mediated cell adhesion assay. Following determination that ASCs preferentially expressed the α5 and β1 monomer units and α5β1 dimer, shear stress was found to significantly increase their expression and resulted in upregulated attachment of the stem cells to surfaces coated with monoclonal antibodies against these proteins. (C) Quantitative polymerase chain reaction demonstrated upregulation of α5 and β1 mRNA expression with maximal expression at 12 and 6 h, respectively. (D) Western blot analysis confirmed upregulation of α5 and β1 protein subunits. Taken together, these results implicate the α5β1 integrin as key in the attachment of ASCs to a fibronectin precoated graft and demonstrate a need for acclimation to a changing shear environment due to their temporal upregulation following stress exposure. *denotes statistical significance p < 0.05.

Article Snippet: Cell integrin blockade Prior to seeding within the tissue culture wells, the ASCs were pretreated with a human monoclonal antibody against the α 5 β 1 integrin (MAB 1969; Chemicon International–Millipore, Billerica, MA) at a concentration of 25 μL/mL for 1 h. Quantitative polymerase chain reaction Total RNA was isolated from ASCs using RNeasy Mini Spin Columns (Qiagen, Valencia, CA).

Techniques: Expressing, Shear, Cell Adhesion Assay, Bioprocessing, Real-time Polymerase Chain Reaction, Western Blot

Journal: ChemistryOpen

Article Title: Insights into the Binding of Cyclic RGD Peptidomimetics to α 5 β 1 Integrin by using Live‐Cell NMR And Computational Studies

doi: 10.1002/open.201600112

Figure Lengend Snippet: Docking best poses of a) compound 7 (green) and b) compound 6 (green) in the crystal structure of the extracellular domain of α 5 β 1 integrin (α 5 subunit pink, β 1 subunit cyan, model from 3VI4.pdb). Only selected integrin residues involved in interactions with the ligand are shown. Non polar hydrogens are hidden for clarity, whereas intermolecular hydrogen bonds are shown as dashed lines.

Article Snippet: Purified recombinant human integrin α 5 β 1 (R&D Systems, Inc., Minneapolis, MN, USA) was diluted to 0.5 μg mL −1 in coating buffer containing 20 m m Tris‐HCl (pH 7.4), 150 m m NaCl, 1 m m MnCl 2 , 2 m m CaCl 2 , and 1 m m MgCl 2 .

Techniques:

Journal: ChemistryOpen

Article Title: Insights into the Binding of Cyclic RGD Peptidomimetics to α 5 β 1 Integrin by using Live‐Cell NMR And Computational Studies

doi: 10.1002/open.201600112

Figure Lengend Snippet: Docking binding modes: a) A and b) B of compound 3 (green) in the crystal structure of the extracellular domain of α 5 β 1 integrin (α 5 subunit pink, β 1 subunit cyan, model from 3VI4.pdb). Only selected integrin residues involved in interactions with the ligand are shown. Non polar hydrogens are hidden for clarity, whereas intermolecular hydrogen bonds are shown as dashed lines.

Article Snippet: Purified recombinant human integrin α 5 β 1 (R&D Systems, Inc., Minneapolis, MN, USA) was diluted to 0.5 μg mL −1 in coating buffer containing 20 m m Tris‐HCl (pH 7.4), 150 m m NaCl, 1 m m MnCl 2 , 2 m m CaCl 2 , and 1 m m MgCl 2 .

Techniques: Binding Assay

Journal: ChemistryOpen

Article Title: Insights into the Binding of Cyclic RGD Peptidomimetics to α 5 β 1 Integrin by using Live‐Cell NMR And Computational Studies

doi: 10.1002/open.201600112

Figure Lengend Snippet: Inhibition of biotinylated fibronectin binding to α 5 β 1 integrin compared with inhibition of biotinylated vitronectin binding to α v β 3 .

Article Snippet: Purified recombinant human integrin α 5 β 1 (R&D Systems, Inc., Minneapolis, MN, USA) was diluted to 0.5 μg mL −1 in coating buffer containing 20 m m Tris‐HCl (pH 7.4), 150 m m NaCl, 1 m m MnCl 2 , 2 m m CaCl 2 , and 1 m m MgCl 2 .

Techniques: Inhibition, Binding Assay

Journal:

Article Title: The EphA8 Receptor Regulates Integrin Activity through p110? Phosphatidylinositol-3 Kinase in a Tyrosine Kinase Activity-Independent Manner

doi: 10.1128/MCB.21.14.4579-4597.2001

Figure Lengend Snippet: (A) Effects of anti-α5β1 and anti-β3 integrin blocking antibodies (Ab) on NIH 3T3 cell adhesion to fibronectin. NIH 3T3 fibroblasts stably expressing wild-type or kinase-inactive EphA8 were detached and incubated for 5 min with either anti-α5β1 or anti-β3 integrin blocking antibody (5 μg/ml). The cells were then replated on fibronectin-coated coverslips and allowed to adhere for 15 min. Data from three independent experiments are presented as means ± SE. (B) Induction of wild-type EphA8 protein expression in NIH 3T3 fibroblasts. Cells were incubated with 2 μg of doxycycline (Dox) per ml for the indicated periods of time and then lysed, and proteins from the lysates were immunoprecipitated with anti-EphA8 antibody. Immunoprecipitates were separated by 7.5% SDS-PAGE and immunoblotted with anti-EphA8 antibody (top); the same blot was stripped of antibodies and reprobed with antiphosphotyrosine antibody (bottom). (C) Effects of anti-α5β1 and anti-β3 integrin blocking antibodies on NIH 3T3 cell attachment stimulated by the induced expression of EphA8. Cells were treated with doxycycline for the indicated periods of time, and cell attachment assays were performed after incubation with either anti-α5β1 or anti-β3 blocking antibodies as described above. (D) Analysis of α5β1 and β3 integrins expressed in NIH 3T3 cells, concomitant with the induced expression of EphA8. EphA8 expression was induced for the indicated times, then the cells were harvested and cell surface proteins were biotinylated; integrins were immunoprecipitated with anti-α5 polyclonal antibody, and biotinylated integrins were detected using streptavidin-HRP (top). Cells were lysed in PLC lysis buffer, and protein concentrations were equalized (bottom). Cell lysates were fractionated by SDS-PAGE and then analyzed by immunobloting using anti-β3 antibody as a probe.

Article Snippet: Monoclonal anti-human integrin α 5 β 1 (JBS5) and murine α 5 β 1 (BMA5) and polyclonal anti-murine integrin α 5 antibodies were from Chemicon.

Techniques: Blocking Assay, Stable Transfection, Expressing, Incubation, Immunoprecipitation, SDS Page, Cell Attachment Assay, Lysis, Western Blot

Journal:

Article Title: The EphA8 Receptor Regulates Integrin Activity through p110? Phosphatidylinositol-3 Kinase in a Tyrosine Kinase Activity-Independent Manner

doi: 10.1128/MCB.21.14.4579-4597.2001

Figure Lengend Snippet: (A) Induction of wild-type EphA8 protein expression in HEK293 epithelial cells. Cells were incubated with 2 μg of doxycycline (Dox) per ml for the indicated times. Proteins from cell lysates were immmunoprecipitated with anti-EphA8 antibody, then separated by 7.5% SDS-PAGE, and immunoblotted with the same antibody (top); the same blot was stripped and then reprobed with antiphosphotyrosine antibody (middle); Bottom, analysis of α5β1 integrin expressed in HEK293 cells, concomitant with the induced expression of EphA8. EphA8 expression was induced for the indicated times, then the cells were harvested, and cell surface proteins were biotinylated. Labeled integrins were immunoprecipitated with anti-α5β1 monoclonal antibody and then detected with streptavidin-HRP. (B) Effect of anti-α5β1 integrin blocking antibodies (Ab) on the cell attachment stimulated by the induced expression of EphA8 in HEK293 cells. The cells were treated with doxycycline for the indicated times, and then cell attachment assays were performed after incubation with anti-α5β1 blocking antibodies as described in the legend to Fig. ​Fig.2A.2A. Note that HEK293 cells were allowed to adhere for 30 min before being washed to remove nonadherent cells, and then incubation was continued for 90 min. The percentage of cells attached after 30 min is shown. Data from three independent experiments are presented as means ± SE. (C) Induction of kinase-inactive EphA8 protein expression in HEK293 epithelial cells. As a positive control, HEK293 cells inducibly expressing the wild-type EphA8 protein were treated with doxycycline for 12 h. Proteins from cell lysates were immmunoprecipitated with anti-EphA8 antibody, then separated by 7.5% SDS-PAGE, and immunoblotted with anti-EphA8 antibody (top); the same blot was stripped and then reprobed with antiphosphotyrosine antibody (bottom). (D) Effects of anti-α5β1 integrin blocking antibodies on cell attachment stimulated by the induced expression of kinase-inactive EphA8 receptor in HEK293 cells. Cell attachment assays were performed as described for panel B.

Article Snippet: Monoclonal anti-human integrin α 5 β 1 (JBS5) and murine α 5 β 1 (BMA5) and polyclonal anti-murine integrin α 5 antibodies were from Chemicon.

Techniques: Expressing, Incubation, SDS Page, Labeling, Immunoprecipitation, Blocking Assay, Cell Attachment Assay, Positive Control

Journal:

Article Title: The EphA8 Receptor Regulates Integrin Activity through p110? Phosphatidylinositol-3 Kinase in a Tyrosine Kinase Activity-Independent Manner

doi: 10.1128/MCB.21.14.4579-4597.2001

Figure Lengend Snippet: The JM deletion EphA8 mutant suppresses EphA8-promoted p110γ PI 3-kinase activity and integrin activation by inhibiting the association of EphA8 with p110γ. HEK293 cells expressing EphA8 in response to doxycycline (Dox) were transiently transfected with the indicated constructs; 12 h after transfection, EphA8 protein expression was induced for 12 h. (A) Cell attachment assays performed as described in previous figure legends. (B) Proteins from cell lysates were immunoprecipitated with anti-p110γ antibody, and then PI-3 kinase activity was measured as described in previous figure legends. (C) Proteins from cell lysates were immunoprecipitated with anti-p110γ antibody and then analyzed by immunoblotting with anti-EphA8 antibody as a probe (top); the same blot was stripped and reprobed with anti-HA and anti-p110γ antibodies (middle and bottom, respectively). (D) The EphA8 JM is sufficient for association with p110γ. A FLAG-tagged murine p110γ construct was transiently transfected into parental cells (lane 5) or HEK293 cells expressing EphA8 in response to doxycycline treatment (lanes 1 to 4). As a control, the FLAG-tagged p110γ protein transiently expressed in parental HEK293 cells was directly immunoprecipitated with anti-FLAG antibody (lane 5). The induction of EphA8 began 12 h after transfection and continued for 24 h (lanes 2 and 4). Proteins from each cell lysate were mixed with approximately equal amounts of purified GST or with GST-JM fusion protein bound to glutathione-Sepharose beads (lanes 1 to 4). The washed beads were separated by 7.5% SDS-PAGE and Western blotted using anti-FLAG antibody as a probe. (E) The EphA8 JM is sufficient for direct association with p110γ. HEK293 cells expressing both exogenous p110γ and wild-type EphA8 were prepared as described for panel D. FLAG-tagged p110γ was directly immunoprecipitated with anti-FLAG antibody, and then the washed beads were directly separated by 7.5% SDS-PAGE and transferred to membranes. The blots were probed with 32P-labeled GST-JM protein to detect p110γ. The labeling of GST-JM protein was performed in an in vitro kinase assay with [γ-32P]ATP and anti-HA immunoprecipitates containing wild-type EphA8 as previously described. Note that the GST-JM fusion protein contained Tyr-615, which is a major phosphorylation site.

Article Snippet: Monoclonal anti-human integrin α 5 β 1 (JBS5) and murine α 5 β 1 (BMA5) and polyclonal anti-murine integrin α 5 antibodies were from Chemicon.

Techniques: Mutagenesis, Activity Assay, Activation Assay, Expressing, Transfection, Construct, Cell Attachment Assay, Immunoprecipitation, Western Blot, Purification, SDS Page, Labeling, In Vitro, Kinase Assay

Journal:

Article Title: A novel integrin ? 5 ? 1 binding domain in module 4 of connective tissue growth factor (CCN2/CTGF) promotes adhesion and migration of activated pancreatic stellate cells

doi: 10.1136/gut.2005.079178

Figure Lengend Snippet: Figure 2 Connective tissue growth factor (CCN2) dependent pancreatic stellate cell (PSC) adhesion is mediated by interactions of module 4 with integrin α5β1. (A) Microtitre wells were precoated at 4°C for 16 hours with phosphate buffered saline (PBS) or 2 μg/ml CCN21–4, CCN23–4, CCN24, or fibronectin (FN) and then blocked with PBS containing 1% bovine serum albumin (BSA) for one hour. Rat activated PSC (2.5×105 cells/ml) were preincubated in serum free medium for 30 minutes in vehicle buffer (no add) or EDTA (5 mM) prior to addition to individual wells at 50 μl/well. After incubation at 37°C for 20 minutes, adherent cells were washed, fixed, and stained by CyQUANT GR dye and quantified by measuring fluorescence intensity at an excitation of 485 nm and an emission of 530 nm. (B) PSC adhesion assays were performed using CCN24 following preincubation of the cells for 30 minutes with EDTA (5 mM) or with addition of Ca2+ (10 mM) or Mg2+ (10 mM) either alone or in combination. (C) PSC were preincubated with 25 μg/ml anti‐integrin α5 or anti‐integrin β1 monoclonal antibodies for 30 minutes prior to adding the cells to the wells that had been precoated with CCN24 (2 μg/ml), FN (2 μg/ml), or vitronectin (VN 4 μg/ml). (D) Microtitre wells were coated with CCN24, FN, or VN, as indicated, above prior to addition of PSC that had been preincubated at 37°C for 30 minutes with vehicle buffer (no add), 25 μg/ml monoclonal anti‐α5β1, or 25 μg/ml normal mouse IgG. Data are means (SD) of quadruplicate determinations and are representative of three experiments. **p<0.01 versus control; ††p<0.01 versus “no add” group.

Article Snippet: Purified human integrin α 5 β 1 , mouse monoclonal anti‐α 5 , anti‐β 1 , rat monoclonal anti‐α 5 β 1 blocking antibodies, and goat polyclonal anti‐α 5 β 1 antibody were purchased from Chemicon Inc. (Temicula, California, USA).

Techniques: Incubation, Staining, CyQUANT Assay, Fluorescence

Journal:

Article Title: A novel integrin ? 5 ? 1 binding domain in module 4 of connective tissue growth factor (CCN2/CTGF) promotes adhesion and migration of activated pancreatic stellate cells

doi: 10.1136/gut.2005.079178

Figure Lengend Snippet: Figure 5 A synthetic connective tissue growth factor module 4 (CCN24) peptide, GVCTDGR, contains an integrin α5β1 binding site. (A) Cell adhesion assays were performed on 96 well plates that had been coated at 2 μg/ml with CCN24, P5 (a synthetic peptide comprising the CVCTDGR, corresponding to residues 285–291 of CCN2) or fibronectin (FN). BSA, bovine serum albumin. (B) Microtitre wells were coated with CCN24 (2 μg/ml) or FN (2 μg/ml) at 4°C for 16 hours and then incubated with 1 μg/ml integrin α5β1 alone or after its preincubation with 35 μM P5 for one hour. CCN24 binding by integrin α5β1 was quantified by ELISA. Data are means (SD) of quadruplicate determinations and are representative of three experiments. *p<0.05, **p<0.01 versus control; †p<0.05, ††p<0.01 versus the “no add” group.

Article Snippet: Purified human integrin α 5 β 1 , mouse monoclonal anti‐α 5 , anti‐β 1 , rat monoclonal anti‐α 5 β 1 blocking antibodies, and goat polyclonal anti‐α 5 β 1 antibody were purchased from Chemicon Inc. (Temicula, California, USA).

Techniques: Binding Assay, Incubation, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: A novel integrin ? 5 ? 1 binding domain in module 4 of connective tissue growth factor (CCN2/CTGF) promotes adhesion and migration of activated pancreatic stellate cells

doi: 10.1136/gut.2005.079178

Figure Lengend Snippet: Figure 6 Connective tissue growth factor module 4 (CCN24) induces integrin α5β1 dependent pancreatic stellate cell (PSC) migration. (A) PSC migration assays were performed by placing the cells in culture inserts (2.5×105 cells/insert) followed by incubation in a 12 well companion plate for six hours in the absence or presence of desired concentrations of CCN24 in the lower chamber. (B) PSC migration assays were performed in the presence of 100 ng/ml of CCN21–4, CCN23–4, CCN24, or 100 ng/ml fibronectin (FN) in the lower chamber. (C) Cell migration assays were performed following 30 minutes of preincubation of PSC with P5 (35 μM), anti‐α5β1 (25 μg/ml), mouse IgG (25 μg/ml), or heparin (2 μg/ml). Data are means (SD) of quadruplicate determinations and are representative of three experiments. **p<0.01 versus control; ††p<0.01 versus the “no add” group.

Article Snippet: Purified human integrin α 5 β 1 , mouse monoclonal anti‐α 5 , anti‐β 1 , rat monoclonal anti‐α 5 β 1 blocking antibodies, and goat polyclonal anti‐α 5 β 1 antibody were purchased from Chemicon Inc. (Temicula, California, USA).

Techniques: Migration, Incubation