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: Biophysical Journal

Article Title: Distinct Binding Interactions of α 5 β 1 -Integrin and Proteoglycans with Fibronectin

doi: 10.1016/j.bpj.2019.07.002

Figure Lengend Snippet: A schematic diagram of energy landscapes for protein unbinding. Two energy minima ( upper curve ) were observed for the fibronectin– α 5 β 1 -integrin interaction, but only one minimum ( lower curve ) was observed for fibronectin with the two PGs studied. ΔG represents the height of a barrier of width χ B . Here, (i) and (o) indicate the inner and outer barriers. Both curves have the same energy at large separations, so they are shifted for clarity. To see this figure in color, go online

Article Snippet: Recombinant human decorin, recombinant human α 5 β 1 -integrin, and recombinant human SDC4 were purchased from R&D Systems (Minneapolis, MN).

Techniques:

Journal: Biophysical Journal

Article Title: Distinct Binding Interactions of α 5 β 1 -Integrin and Proteoglycans with Fibronectin

doi: 10.1016/j.bpj.2019.07.002

Figure Lengend Snippet: Representative force spectroscopy data for the unbinding of SDC4 ( top ), decorin ( middle ), and α 5 β 1 -integrin ( bottom ) from a fibronectin-immobilized surface. The retraction speeds for these data are 2, 1, and 3 μ m/s, respectively. For clarity, the data for SDC4 and decorin are offset by 200 and 100 pN, respectively. To see this figure in color, go online

Article Snippet: Recombinant human decorin, recombinant human α 5 β 1 -integrin, and recombinant human SDC4 were purchased from R&D Systems (Minneapolis, MN).

Techniques: Force Spectroscopy

Journal: Biophysical Journal

Article Title: Distinct Binding Interactions of α 5 β 1 -Integrin and Proteoglycans with Fibronectin

doi: 10.1016/j.bpj.2019.07.002

Figure Lengend Snippet: Frequency distributions for extracted rupture forces, taken at different velocities for the unbinding of fibronectin from α 5 β 1 -integrin ( A ), SDC4 ( B ), and decorin ( C ). The number of measurements, n , from which the distributions were obtained, is shown in each panel. The fits (shown) are to either Gaussian or log-Gaussian models, and the modal averages of the rupture force for the corresponding loading rate were obtained. (These may be distinguished by noting that the fit to a log-Gaussian model passes through the origin.) Included errors correspond to the width of the 95% confidence interval for the fit. Standard error values for each fit, provided by statistical software package GraphPad, were significantly smaller than the error values (95% confidence interval width) quoted here. The log-Gaussian fit was used when the Gaussian fit was unsatisfactory, although the modal average is model independent. To see this figure in color, go online.

Article Snippet: Recombinant human decorin, recombinant human α 5 β 1 -integrin, and recombinant human SDC4 were purchased from R&D Systems (Minneapolis, MN).

Techniques: Software

Journal: Biophysical Journal

Article Title: Distinct Binding Interactions of α 5 β 1 -Integrin and Proteoglycans with Fibronectin

doi: 10.1016/j.bpj.2019.07.002

Figure Lengend Snippet: The dynamic force spectrum describing the unbinding between fibronectin and α 5 β 1 -integrin ( A ), SDC4 ( B ), and decorin ( C ). The linear increase of the average rupture force with the logarithm of the average loading rate for each pulling velocity, fitted using the Bell-Evans relation , is shown. Included errors correspond to the width of the 95% confidence interval from which average rupture forces were extracted. Two energy barriers were observed for the unbinding of fibronectin from α 5 β 1 -integrin.

Article Snippet: Recombinant human decorin, recombinant human α 5 β 1 -integrin, and recombinant human SDC4 were purchased from R&D Systems (Minneapolis, MN).

Techniques:

Journal: Biophysical Journal

Article Title: Distinct Binding Interactions of α 5 β 1 -Integrin and Proteoglycans with Fibronectin

doi: 10.1016/j.bpj.2019.07.002

Figure Lengend Snippet: Extracted Energetics for Each Energy Barrier Revealed in the Dynamic Spectra for the Unbinding of Fibronectin from Both the Inner, I, and Outer, o, Barriers of α 5 β 1 -Integrin, as well as Those for SDC4 and Decorin

Article Snippet: Recombinant human decorin, recombinant human α 5 β 1 -integrin, and recombinant human SDC4 were purchased from R&D Systems (Minneapolis, MN).

Techniques:

Journal: Oncogene

Article Title: C1GALT1 high expression is associated with poor survival of patients with pancreatic ductal adenocarcinoma and promotes cell invasiveness through integrin α v

doi: 10.1038/s41388-020-01594-4

Figure Lengend Snippet: A Effects of C1GALT1 knockdown on cell spreading in HAPF-II and HPAC cells. Pancreatic cancer cells were treated with control (siControl) or C1GALT1 siRNA (siC1GALT1-3). Representative images showing amplified cells in one fourth of a field. Scale bars, 20 μm. Spreading cells were quantified under a phase-contrast microscope and are shown in the lower panel. * p < 0.05 by student’s t test. B C1GALT1 knockdown inhibited cell-extracellular matrix (ECM) adhesion. Cells were plated onto 96-well plates coated with 2.5 μg/μL of bovine serum albumin (BSA), collagen I (Col I), collagen IV (Col IV), fibronectin (FN), or laminin (LAM). Specific ECM-adhered cells were calculated by subtracting BSA-adhered cells. Results are presented as mean ± SD of six independent experiments. ** p < 0.01; *** p < 0.001 by student’s t test. C Effects of C1GALT1 knockdown on tyrosine phosphorylation of FAK. HPAF-II and HPAC cells were plated onto culture plates coated with 1 µg/mL of different ECM proteins, as indicated, in serum-free DMEM for 3 h. Changes in FAK phosphorylation at Y397 and Y925 were analyzed by Western blotting. GAPDH was used as an internal loading control. D Effects of C1GALT1 knockdown on Tn antigen expression of selected integrins, including α v , α 5 , β 1 , α 2 , and α 3. Changes in Tn expression were analyzed using VVA pull-down (PD) assays in C1GALT1 knockdown HAPF-II and HPAC cells. Proteins were detected by Western blot (WB) analysis. GAPDH was used as an internal loading control.

Article Snippet: Functional blocking antibodies for integrin β 1 (P4C10), integrin α 5 β 1 (JBS5), integrin α 5 (P1D6), and integrin α v (AV1) were purchased from Merck KGaA.

Techniques: Amplification, Microscopy, Western Blot, Expressing

Journal: Oncogene

Article Title: C1GALT1 high expression is associated with poor survival of patients with pancreatic ductal adenocarcinoma and promotes cell invasiveness through integrin α v

doi: 10.1038/s41388-020-01594-4

Figure Lengend Snippet: A Western blots showing C1GALT1 stable knockdown in HPAF-II and HPAC cells and C1GALT1 overexpression in MIAPaca2 cells. PDAC cells were transfected using lentivirus-mediated C1GALT1 shRNA in pLKO.1 vector (shC1GALT1) compared with its empty vector (shControl) and C1GALT1 was overexpressed using C1GALT1/pcDNA3.1 plasmid (C1GALT1) compared with its empty plasmid (mock). GAPDH was used as an internal loading control. B C1GALT1 knockdown or overexpression did not alter the expression of surface integrins β 1 , α 5 , α 5 β 1 , and α v in HAPF-II and HPAC cells analyzed by flow cytometry. Unstained cells were used as a negative control (-). C Effects of functional blocking antibodies against integrins on PDAC cell invasion. C1GALT1 knockdown HPAF-II and HPAC cells and C1GALT1 overexpressing MIAPaca2 cells were subjected to Matrigel invasion assays. Cells were treated with 10 μg/mL of blocking antibody, as indicated. IgG was used as a control. Invasion of HPAF-II, HPAC, and MIAPaca2 cells was analyzed after 24 and 48 h. Results are presented as mean ± SD of four independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001 by student’s t test. D Effects of functional blocking antibodies on FAK phosphorylation in PDAC cells using Western blotting. Functional blocking antibody against integrin α v or α 5 was used, as indicated, to treat HPAF-II and HPAC cells for 30 min before seeding to culture plates. IgG was used as a control. GAPDH was used as an internal loading control. E Western blots showing changes in Tn antigens on cell surface integrins α V and α 5 . Cells were surface biotinylated. Plasma Membrane Protein Extraction Kit (Abcam) was used for extraction and purification of plasma membrane proteins from HPAF-II and HPAC cells. VVA pull-down (PD) assays were performed to assess changes in Tn antigens on integrins. Proteins were detected by Western blot (WB) analysis. F A schematic diagram illustrating the proposed mechanism by which C1GALT1 promotes tumor growth and metastasis in pancreatic cancer. C1GALT1 modifies O-glycans on integrins, including α 5 , α v , and β 1 , which leads to altered integrin-FAK signaling. Integrin α v (red color) is proposed to play a critical role in C1GALT1-mediated invasiveness. This pathway is coordinated with other C1GALT1-regulated pathways, such as receptor tyrosine kinases (RTKs) and mucins, to promote tumor growth and metastasis in pancreatic cancer. FN fibronectin, VN vitronectin.

Article Snippet: Functional blocking antibodies for integrin β 1 (P4C10), integrin α 5 β 1 (JBS5), integrin α 5 (P1D6), and integrin α v (AV1) were purchased from Merck KGaA.

Techniques: Western Blot, Over Expression, Transfection, shRNA, Plasmid Preparation, Expressing, Flow Cytometry, Negative Control, Functional Assay, Blocking Assay, Protein Extraction, Purification

Journal: Perspectives in Medicinal Chemistry

Article Title: The Role of Integrins in Cancer and the Development of Anti-Integrin Therapeutic Agents for Cancer Therapy

doi:

Figure Lengend Snippet: Integrin inhibitors in clinical development as anticancer agents.

Article Snippet: M200 , Volociximab Eos-200-4 , α 5 β 1 , Currently in phase II , Protein design Labs , Protein Design Labs, www.pdl.com.

Techniques:

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: 13 Briefly, microtitre wells (Dynex Technology, Chantilly, Virginia, USA) were precoated with CCN2 proteins or FN and, after extensive blocking and washing, were incubated with 1 μg/ml integrin α 5 β 1 .

Techniques: Saline, Incubation, Staining, CyQUANT Assay, Fluorescence, Bioprocessing, Control

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 3 Role of cell surface heparan sulphate proteoglycan in connective tissue growth factor module 4 (CCN24) mediated pancreatic stellate cell (PSC) adhesion. (A) Cell adhesion assays were performed on CCN24 precoated microtitre wells using PSC that were treated with vehicle buffer (no add) or heparin (2 μg/ml) prior to plating. (B) Microtitre wells were coated with 2 μg/ml CCN24 or fibronectin (FN) prior to addition of PSC that had been pretreated at 37°C for 30 minutes with vehicle buffer (no add), 2 units/ml heparinase I, or chondroitinase ABC. (C) PSC were cultured in complete medium containing 10 mM NaClO3 for 48 hours in the presence or absence of 10 mM Na2SO4 prior to addition to CCN24 precoated wells. 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: 13 Briefly, microtitre wells (Dynex Technology, Chantilly, Virginia, USA) were precoated with CCN2 proteins or FN and, after extensive blocking and washing, were incubated with 1 μg/ml integrin α 5 β 1 .

Techniques: Cell Culture, Control

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: 13 Briefly, microtitre wells (Dynex Technology, Chantilly, Virginia, USA) were precoated with CCN2 proteins or FN and, after extensive blocking and washing, were incubated with 1 μg/ml integrin α 5 β 1 .

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