function blocking antibodies β 1 integrin  (Becton Dickinson)

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: Cell Death and Differentiation

Article Title: A vitronectin-derived peptide reverses ovariectomy-induced bone loss via regulation of osteoblast and osteoclast differentiation

doi: 10.1038/cdd.2017.153

Figure Lengend Snippet: VnP-16 promotes osteogenic cell attachment through direct interaction with β1 integrin. (a) Attachment of human osteogenic cells pretreated with EDTA (5 mM), MnCl2 (500 μM), or heparin (100 μg/ml) to VnP-16. The cells were seeded onto plates that were precoated with VnP-16 (9.1 μg/cm2) for 1 h. (b) The effects of various integrin-blocking antibodies on cell attachment to VnP-16. (c–e) Immunoblotting (c) and densitometric analysis (d) of β1 integrin, and cell attachment to VnP-16 (e) in osteogenic cells that were transfected with a control (Con) or β1 integrin-specific siRNA (10 nM; β1 integrin). (f) Streptavidin-bead pulldown assay with the biotinylated SP or biotinylated VnP-16 peptides from extracts of osteogenic cells that were cultured on biotinylated SP- or biotinylated VnP-16-coated dishes for 30 min. Data in (a,b,e) (n=4), and (d) (n=2) represent the mean±SD. **P<0.01

Article Snippet: The cells (5 × 10 4 cells/250 μ l) were preincubated with 5 mM EDTA, 500 μ M MnCl 2 , 100 μ g/ml heparin, and 10 μ g/ml function-blocking antibodies against the integrin subunits α 1 (FB12), α 2 (P1E6), α 3 (P1B5), α 4 (P4C2), α 5 (P1D6), α 6 (NKI-GoH3), β 3 (B3A; Chemicon, Temecula, CA, USA), α v (AV1), or β 1 (6S6; Millipore) for 15 min at 37 °C.

Techniques: Cell Attachment Assay, Blocking Assay, Western Blot, Transfection, Control, Cell Culture

Journal: Cell Death and Differentiation

Article Title: A vitronectin-derived peptide reverses ovariectomy-induced bone loss via regulation of osteoblast and osteoclast differentiation

doi: 10.1038/cdd.2017.153

Figure Lengend Snippet: VnP-16 promotes osteogenic differentiation through β1 integrin/FAK signaling. (a,b) Immunoblotting (a) and densitometric analyses (b) of phospho-FAK, phospho-Akt Ser473, phospho-PKCδ Thr505, and phospho-c-Src Tyr416 in osteogenic cells that were cultured for 3 h on plates coated with vitronectin (0.23 μg/cm2), SP, or VnP-16 (9.1 μg/cm2). (c,d) Immunoblotting (c) and densitometric analyses (d) of total FAK (t-FAK) and phospho-FAK Tyr397 in osteogenic cells that were pretreated with PF-573228 for 1 h. (e) Attachment of cells that were treated with PF-573228 for 1 h in serum-free medium to plates precoated with VnP-16 (9.1 μg/cm2). (f) The effects of VnP-16 on alkaline phosphatase activity and calcium deposition in SKP-derived mesenchymal precursors (MPs), mouse calvarial osteoblast precursors (MC3T3-E1), and human osteogenic cells (Osteogenic). The cells were cultured in osteogenic differentiation medium containing VnP-16 or SP (50 μg/0.5 ml) for 2 weeks. (g) The effects of PF-573228 on alkaline phosphatase activity and calcium deposition in SKP-derived mesenchymal precursors, MC3T3-E1, and human osteogenic cells. The cells were cultured on VnP-16-treated (9.1 μg/cm2) plates in osteogenic differentiation medium with or without 1 μM PF-573228 for 2 weeks. (h–j) Immunoblotting (h) and densitometric analyses (i) of t-FAK, and dose-dependent attachment (j) of control or FAK-specific siRNA-treated (100 nM) osteogenic cells to VnP-16. (k) Determination of apoptotic cells in osteogenic cells that were cultured for 24, 48, and 96 h on plates coated with SP or VnP-16 (9.1 μg/cm2) by TUNEL assay. Data in (b, d and i) (n=3), and (e, j and k) (n=4) represent the mean±SD. *P<0.05 or **P<0.01 compared to vehicle or control siRNA

Article Snippet: The cells (5 × 10 4 cells/250 μ l) were preincubated with 5 mM EDTA, 500 μ M MnCl 2 , 100 μ g/ml heparin, and 10 μ g/ml function-blocking antibodies against the integrin subunits α 1 (FB12), α 2 (P1E6), α 3 (P1B5), α 4 (P4C2), α 5 (P1D6), α 6 (NKI-GoH3), β 3 (B3A; Chemicon, Temecula, CA, USA), α v (AV1), or β 1 (6S6; Millipore) for 15 min at 37 °C.

Techniques: Western Blot, Cell Culture, Activity Assay, Derivative Assay, Control, TUNEL Assay

Journal: Scientific Reports

Article Title: S100A4 contributes to colitis development by increasing the adherence of Citrobacter rodentium in intestinal epithelial cells

doi: 10.1038/s41598-017-12256-z

Figure Lengend Snippet: S100A4 increases C. rodentium adherence to CT26 cells. ( A ) FISH analysis of conventional WT and S100A4 −/− mice colon on day 0 and day 7. Colon tissues were probed with a universal bacterial FISH probe (green) and counterstained with DAPI (blue), (scale bars, 20 μm). Representative images of the distal colon were shown ( n = 3). ( B ) Real-time quantitative PCR analysis of the expression of mRNA encoding adhesion molecules in WT and S100A4 −/− mice colons on day 7 p.i. GAPDH was used as the reference control, ( n = 4); ** P < 0.01. The mRNA level of WT mice is set as 1.00 to calibrate the relative level of S100A4 −/− mice. ( C ) Real-time quantitative PCR analysis of the expression of mRNA encoding adhesion molecules in CT26 cells treated for 0 or 8 h with S100A4. GAPDH was used as the reference control, ( n = 4); ** P < 0.01. The mRNA level of 0 h is set as 1.00 to calibrate the relative level of 8 h. ( D ) Real-time quantitative PCR analysis of the expression of mRNA encoding adhesion molecules in CT26 cells treated for 0 or 8 hours with S100A4 after stimulating with FPS-ZM1 (10 μg/ml) for 1 h. GAPDH was used as the reference control, ( n = 4); * P < 0.05. The mRNA level of cells that are not administrated with inhibitor FPS-ZM1 is set as 1.00 to calibrate the relative level of cells administrated with inhibitor FPS-ZM1. ( E ) The adherent ratios of C. rodentium to a CT26 cell monolayer were quantified by calculating CFU. CT26 cells were pretreated with S100A4 (1000 ng/ml) for 0–8 h before infection with C. rodentium , ( n = 4); * P < 0.05. ( F ) The adherent ratio of C. rodentium to CT26 cells was quantified after stimulating with integrin blocker AIIB2 (2.5 μg/ml) for 1 h and S100A4 for 8 h, ( n = 4); ** P < 0.01. ( G ) Bacterial titers in fecal homogenates from AIIB2 treated WT mice and control WT mice on day 7 after C . rodentium infection ( n = 5); * P < 0.05.

Article Snippet: Integrin β−1 function-blocking antibody AIIB2 was obtained from Millipore (Temecula, CA).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Control, Infection

Journal: PLoS ONE

Article Title: Galectin-8 Promotes Cytoskeletal Rearrangement in Trabecular Meshwork Cells through Activation of Rho Signaling

doi: 10.1371/journal.pone.0044400

Figure Lengend Snippet: Normal human TM cells were incubated on microtiter wells coated with BSA, fibronectin, or Gal8 in DPBS, in the presence and absence of a function-blocking anti-β 1 integrin antibody (JB1A), or control mouse IgG. Following incubation at 37°C for 30 min, cells were fixed and stained with crystal violet. Attached cells in fibronectin-coated wells are set as 100% (positive control); attached cells in other wells are presented as percent of positive control. Data are expressed as mean±SEM and analyzed with one-way ANOVA. * P <0.05 vs IgG; ** P <0.01 vs media or IgG; *** P <0.001 vs media. B and C: Cell spreading assay. TM cells were fixed with 4% paraformaldehyde after adhesion for 30 min. F-actin was stained with rhodamine-labeled phalloidin and cell nuclei were labeled with DAPI. Random fields of each experimental condition were photographed, and spread areas of individual cells were quantified with ImageJ. Representative micrographs of TM cells incubated in the presence and the absence of anti-β 1 integrin antibody are shown in C. Data are presented as Box–whisker plot (after Tukey) and analyzed with one-way ANOVA. *** P <0.001 vs media or IgG. This experiment was performed three times with reproducible results. Bar: 100 µm.

Article Snippet: To assess the involvement of β 1 -integrins in the adhesion of TM cells on Gal8, cells were incubated in the presence of an anti-integrin β 1 functional-blocking antibody, JB1A (1∶50 dilution; 10 min, 25°C; EMD Millipore, Billerica, MA).

Techniques: Incubation, Blocking Assay, Control, Staining, Positive Control, Labeling, Whisker Assay

Journal: Oncogene

Article Title: Ligand independent activation of c-Met by fibronectin and α 5 β 1 -integrin regulates ovarian cancer invasion and metastasis

doi: 10.1038/onc.2010.532

Figure Lengend Snippet: ( a ) HeyA8 cells (1×10 6 ) were injected intraperitoneally (i.p.) in nude mice which were randomized into 2 groups of 10/group receiving 10mg/kg human α 5 β 1 -integrin antibody (Hu α 5 Ab) or control IgG. The effect of Hu α 5 Ab on tumor weight was assessed on day 20. ( b ) SKOV3ip1 cells (1×10 6 ) were similarly injected i.p. treatment started on day 8 and tumor weight and the number of metastases assessed on day 35. Representative images of SKOV3ip1 tumors stained for Ki-67 (x400). Images of sections from 5 mice were quantified (**p<0.01). ( c ) SKOV3ip1 cells (1×10 6 ) were injected i.p. in nude mice randomized into 6 groups of 10 each and were treated with the indicated antibodies starting on day 8. Mice were euthanized on day 35 and the effect on tumor weight and number of metastases was assessed. (*p<0.001, **p<0.01,). ( d ) The effect of co-administering SKOV3ip1 cells with a single dose of Hu α 5 Ab injected i.p. in mice (prevention) was compared with treatment of SKOV3ip1 tumor bearing mice starting 8 days after injection of cancer cells (intervention). The effect of treatment on tumor weight and number of metastases was measured. ( e ) A single dose of murine α 5 Ab was co-injected with SKOV3ip1 cells i.p. in nude mice (prevention) and tumor weight and number of metastases was measured on day 35. ( f ) Kaplan-Meier curve for survival. SKOV3ip1 cells were injected i.p. and 10 mice/group were treated with the human α 5 Ab or control IgG twice a week starting from day 8 (p<0.0001; log-rank test).

Article Snippet: The function blocking antibodies against human α 5 β 1 -integrin (M200, referred to as Hu α 5 Ab), mouse α 5 β 1 -integrin (339.1, referred to as Murine α 5 Ab) and Avastin® (Genentech) were provided by Facet Biotech Corp (Redwood City, CA).

Techniques: Injection, Control, Staining

Journal: Oncogene

Article Title: Ligand independent activation of c-Met by fibronectin and α 5 β 1 -integrin regulates ovarian cancer invasion and metastasis

doi: 10.1038/onc.2010.532

Figure Lengend Snippet: ( a ) Left: HeyA8 cells were transfected with α 5 -integrin siRNA or treated with human α 5 Ab and plated on fibronectin followed by western blots for α 5 -Integrin, p-c-MetY 1230,1234,1235 and c-Met. Right: HeyA8 cells were plated on fibronectin, collagen or poly-L-Lysine and were treated with human α 5 Ab followed by immunoblotting for phospho-c-Met Y 1230,1234,1235 , c-Met and actin. ( b ) HeyA8 cells were plated on fibronectin or poly-L-Lysine and treated with human α 5 Ab for 24 h. Cell lysates were immunoprecipitated (IP) with an antibody against α 5 -integrin or c-Met, respectively followed by immunoblotting (WB) for α 5 -integrin or c-Met. ( c ) Confocal microscopy. HeyA8 cells were plated on fibronectin coated cover slips, detected with c-Met and α 5 -integrin antibodies followed by fluorescein or Alexa Fluor 594 labeled secondary antibodies respectively. ( d ) SKOV3ip1 xenografts were treated with Hu α 5 Ab or IgG as described in Figure 1 a . Left: Western blots for p-c-Met Y 1230,1234,1235 and c-Met using tumor lysates. Right: Image of tumor sections stained for p-c-Met Y 1200,1234,1235 (x200 and x400). Images of sections from 5 mice were quantified using Image J with color deconvolution (*p<0.001). ( e ) Human umbilical vein endothelial cells (HUVEC) were plated on fibronectin and treated with human α 5 Ab followed by western blots for p-c-MetY 1230,1234,1235 and c-Met.

Article Snippet: The function blocking antibodies against human α 5 β 1 -integrin (M200, referred to as Hu α 5 Ab), mouse α 5 β 1 -integrin (339.1, referred to as Murine α 5 Ab) and Avastin® (Genentech) were provided by Facet Biotech Corp (Redwood City, CA).

Techniques: Transfection, Western Blot, Immunoprecipitation, Confocal Microscopy, Labeling, Staining

Journal: Oncogene

Article Title: Ligand independent activation of c-Met by fibronectin and α 5 β 1 -integrin regulates ovarian cancer invasion and metastasis

doi: 10.1038/onc.2010.532

Figure Lengend Snippet: ( a ) Top: HeyA8 cells were transfected with α 5 -integrin siRNA or treated with human α 5 Ab and plated on fibronectin for 24h. Western blots were performed for p-FAK-Y 397 , p-FAK-Y 861 , FAK, and α 5 -integrin. ( b ) HeyA8 cells were transfected with CD2FAK (constitutively active FAK), CD2FAK-Y397F (mutated/inactive FAK) or CD2 (vector). Top : Expression and phosphorylation (Y 397 ) of CD2FAK (160kDa) was verified by western blotting. Bottom: Transfected HeyA8 cells were added to matrigel coated Boyden chamber with human α 5 Ab or IgG and allowed to invade for 24 h. The number of invaded cells was counted. ( c ) HeyA8 cells were plated on fibronectin and treated with human α 5 Ab or IgG for 24 h. Phosphorylated (p)-Src Y 416 and Src was detected by immunoblotting.

Article Snippet: The function blocking antibodies against human α 5 β 1 -integrin (M200, referred to as Hu α 5 Ab), mouse α 5 β 1 -integrin (339.1, referred to as Murine α 5 Ab) and Avastin® (Genentech) were provided by Facet Biotech Corp (Redwood City, CA).

Techniques: Transfection, Western Blot, Plasmid Preparation, Expressing, Phospho-proteomics

Journal: Oncogene

Article Title: Ligand independent activation of c-Met by fibronectin and α 5 β 1 -integrin regulates ovarian cancer invasion and metastasis

doi: 10.1038/onc.2010.532

Figure Lengend Snippet: Proposed role of ligand independent activation of c-Met involving a fibronectin/α 5 β 1 -integrin/c-Met/Src/FAK dependent signaling pathway.

Article Snippet: The function blocking antibodies against human α 5 β 1 -integrin (M200, referred to as Hu α 5 Ab), mouse α 5 β 1 -integrin (339.1, referred to as Murine α 5 Ab) and Avastin® (Genentech) were provided by Facet Biotech Corp (Redwood City, CA).

Techniques: Activation Assay

Journal: British Journal of Cancer

Article Title: Neuropilin-1 antagonism in human carcinoma cells inhibits migration and enhances chemosensitivity

doi: 10.1038/sj.bjc.6605539

Figure Lengend Snippet: Suppression of cell–matrix adhesion and enhancement of the inhibitory effects of an integrin- β 1-function-blocking antibody by EG3287. ( A , C ) A549 and ACHN cells were pretreated for 30 min with the indicated concentrations of EG3287 and seeded to wells coated with the indicated matrix proteins. After 90 min of incubation, attached cells were labelled with calcein-AM and measured using a fluorescence plate reader. * P <0.05; ** P <0.01 vs untreated control. ( B , D ) A549 and ACHN cells were pretreated for 30 min with an integrin- β 1-blocking antibody at the indicated concentrations in the absence or presence of 100 μ M EG3287, and cell adhesion to matrix proteins was measured. * P <0.05; ** P <0.01 for integrin- β 1 antibody alone vs integrin- β 1 antibody plus EG3287.

Article Snippet: A functional blocking antibody against the integrin β 1-subunit was from Millipore (Livingston, UK).

Techniques: Blocking Assay, Incubation, Fluorescence, Control

Journal:

Article Title: COOPERATION OF THE METALLOPROTEASE, DISINTEGRIN, AND CYSTEINE-RICH DOMAINS OF ADAM12 DURING INHIBITION OF MYOGENIC DIFFERENTIATION *

doi: 10.1074/jbc.M413550200

Figure Lengend Snippet: A, The effect of integrin β1 function-blocking antibody on cell adhesion in the presence of Mn2+. Wells of 96-well plates were coated with the DC or X proteins at 10 µg/ml. C2C12 cells were suspended in Tyrode’s buffer with 1 mM Mn2+ and incubated for 15 min without any antibodies (Control, black bars) or with 20 µg/ml of Ha2/5, an integrin β1 function-blocking antibody (grey bars). Cells were then added to the wells (105 cells/well) and incubated for 1 h at 37°C. After removing unbound cells, bound cells were fixed and stained with 0.04% crystal violet. Results are shown as the average from 3 measurements, ±S.E. B, The effect of integrin β1 function-blocking antibody on cell adhesion in the absence of Mn2+. Wells of 96-well plates were coated with the DC or X proteins at 1 µg/ml. Cells were incubated for 15 min in Tyrode’s buffer without 1 mM Mn2+, without antibodies (Control), with 20 µg/ml of Ha2/5 antibody (grey bars), with 20 µg/ml of RMV-7, an integrin αvβ3 function-blocking antibody (white bars), or with 20 µg/ml of Ha2/5 and 20 µg/ml of RMV-7 antibodies (hatched bars) prior to adhesion assays. C, Adhesion to the X domain does not support cell survival. Wells of 96-well plates were coated with 10 µg/ml of laminin (L), 20 µg/ml of the X domain of ADAM12, 10 µg/ml of laminin and 20 µg/ml of X (L+X), or 20 µg/ml of polylysine (P). C2C12 were suspended in Tyrode’s buffer and added to the wells (105 cells/well). After 1 h, unbound cells were removed, and serum-free DMEM medium was added to the wells. The number of viable cells was determined right after adding DMEM to cells (time 0 h, white bars) or after 24 h (black bars). Results are shown as the average from four measurements, ±S.E.

Article Snippet: In some experiments, cells were incubated with 1 mM MnCl 2 , integrin β 1 function-blocking antibody Ha2/5 (Pharmingen), or integrin α v β 3 function-blocking antibody RMV-7 (Chemicon) at room temperature for 15 min prior to adding to wells.

Techniques: Blocking Assay, Incubation, Staining