Journal: Journal of Virology

Article Title: Roles of the Putative Integrin-Binding Motif of the Human Metapneumovirus Fusion (F) Protein in Cell-Cell Fusion, Viral Infectivity, and Pathogenesis

doi: 10.1128/JVI.03491-13

Figure Lengend Snippet: α5β1 and αv integrin-specific antibodies block cell-cell fusion triggered by hMPV F protein. (A) Syncytium formation induced by hMPV F protein expression in the presence of integrin antibodies. Vero E6 cells in 48-well plates were transfected with 0.8 μg of pCAGGS-F or pCAGGS. After incubation with a plasmid-Lipofectamine mixture for 8 h, cells continued to grow in Opti-MEM containing 10 μg/ml of integrin antibody and 0.2 μg/ml of TPCK-trypsin. At 48 h, monolayers were fixed with methanol and stained with Giemsa. Syncytia are indicated by arrows. (B) Content-mixing fusion assay in the presence of integrin antibodies. Vero E6 cells were cotransfected with 2 μg of pCAGGS-F and a reporter gene plasmid (pGINT7). At 24 h posttransfection, the cells were detached with trypsin and mixed with equal numbers of BHK-SR19-T7 cells, which express T7 RNA polymerase. Then, the cells were incubated with 2 ml of Opti-MEM containing 5 μg/ml of selected integrin antibody and 0.2 μg/ml of TPCK-trypsin for 12 h. The cells were lysed and mixed with an equal amount of the β-galactosidase substrate chlorophenol red-β-d-galactopyranoside (16 mM). The extent of fusion was quantitated by use of a microplate spectrophotometer at an absorbance of 570 nm. Percent fusion for each antibody treatment was normalized to the fusion of pCAGGS-F in the absence of integrin antibody. The data shown are averages for three independent experiments.

Article Snippet: Twenty picomoles of synthetic small interfering RNAs (siRNAs) targeting either human integrin subtype α5 (sc-29372; Santa Cruz Biotechnology, CA) or αv (sc-29373; Santa Cruz Biotechnology) or control siRNA (sc-37007; Santa Cruz Biotechnology) was transfected into Vero E6 cells (at a confluence of 75%) in 24-well plates using Oligofectamine reagents (Invitrogen) according to the manufacturer's instructions.

Techniques: Blocking Assay, Expressing, Transfection, Incubation, Plasmid Preparation, Staining, Single Vesicle Fusion Assay, Spectrophotometry

Journal: Journal of Virology

Article Title: Roles of the Putative Integrin-Binding Motif of the Human Metapneumovirus Fusion (F) Protein in Cell-Cell Fusion, Viral Infectivity, and Pathogenesis

doi: 10.1128/JVI.03491-13

Figure Lengend Snippet: siRNAs targeting α5 and αv back cell-cell fusion triggered by hMPV F protein. (A) Knockdown of integrin α5 and αv expression by siRNA. Twenty picomoles of synthetic siRNA targeting human integrin subtype α5 or αv as well as control siRNA was transfected into Vero E6 cells in 24-well plates using Oligofectamine reagents according to the manufacturer's instructions. After 48 h posttransfection, the expression of α5 or αv was detected by Western blotting. (B) Syncytium formation induced by F protein of hMPV after knockdown of integrins α5 and αv. Vero E6 cells in 24-well plates were transfected with 20 pmol of synthetic siRNA targeting human integrin subtype α5 or αv as well as control siRNA. After treatment with siRNAs for 24 h, Vero E6 cells were transfected with 0.8 μg of pCAGGS-F using Lipofectamine Plus reagents and then subjected to pH pulses (pH 5.0). At 48 h, monolayers were fixed with methanol and stained with Giemsa. (C) Quantitation of syncytium formation after knockdown of integrins α5 and αv. The number of syncytia (≥4 nuclei in each syncytium) was counted under a microscope using six randomly selected fields in each siRNA-treated or untreated well. The mean number of syncytia per field was calculated for each treatment. The percent fusion for each siRNA treatment was normalized by the mean number of syncytia in cells transfected with pCAGGS-F without siRNA treatment. The data shown are averages for three independent experiments.

Article Snippet: Twenty picomoles of synthetic small interfering RNAs (siRNAs) targeting either human integrin subtype α5 (sc-29372; Santa Cruz Biotechnology, CA) or αv (sc-29373; Santa Cruz Biotechnology) or control siRNA (sc-37007; Santa Cruz Biotechnology) was transfected into Vero E6 cells (at a confluence of 75%) in 24-well plates using Oligofectamine reagents (Invitrogen) according to the manufacturer's instructions.

Techniques: Knockdown, Expressing, Control, Transfection, Western Blot, Staining, Quantitation Assay, Microscopy

Journal: Journal of Virology

Article Title: Roles of the Putative Integrin-Binding Motif of the Human Metapneumovirus Fusion (F) Protein in Cell-Cell Fusion, Viral Infectivity, and Pathogenesis

doi: 10.1128/JVI.03491-13

Figure Lengend Snippet: Integrin α5β1and αv antibodies inhibit hMPV infectivity in host cells. Confluent monolayers of LLC-MK2 cells in 96-well plates were pretreated with each integrin antibody (20 μg/ml) at 37°C for 1 h. Then, the cells were shifted to a 4°C incubator for 30 min. The cells were incubated with hMPV at an MOI of 100 PFU/well. After incubation on ice for 1 h (with shaking every 15 min), the inoculum was removed and the cells were washed with cold Opti-MEM 3 times. The infected cells were then incubated with fresh DMEM at 37°C in 5% CO2. After 24 h, the binding and infectivity were determined by counting the number of immunostaining spots. The percent infectivity for each antibody treatment was normalized by the infectivity of hMPV without antibody treatment. The data shown are averages for three independent experiments.

Article Snippet: Twenty picomoles of synthetic small interfering RNAs (siRNAs) targeting either human integrin subtype α5 (sc-29372; Santa Cruz Biotechnology, CA) or αv (sc-29373; Santa Cruz Biotechnology) or control siRNA (sc-37007; Santa Cruz Biotechnology) was transfected into Vero E6 cells (at a confluence of 75%) in 24-well plates using Oligofectamine reagents (Invitrogen) according to the manufacturer's instructions.

Techniques: Infection, Incubation, Binding Assay, Immunostaining

Journal: Journal of Virology

Article Title: Roles of the Putative Integrin-Binding Motif of the Human Metapneumovirus Fusion (F) Protein in Cell-Cell Fusion, Viral Infectivity, and Pathogenesis

doi: 10.1128/JVI.03491-13

Figure Lengend Snippet: Infectivity of hMPV in an integrin-deficient cell line. (A) hMPV has defects in infectivity in an integrin-deficient cell line. Confluent monolayers of GD1286 or GD25 cells in 24-well plates were incubated at 4°C for 30 min. The cells were infected with 100 PFU of hMPV per well. After incubation on ice for 1 h (with shaking every 15 min), the inoculum was removed and the cells were washed with cold DMEM 3 times. The infected cells were then incubated with fresh medium at 37°C in 5% CO2. After 24 h, the binding capacity was determined by counting the number of immunostaining spots. Percent infectivity in GD25 cells was normalized by the infectivity of hMPV in GD1286 cells. The data shown are averages for three independent experiments. (B) hMPV forms much smaller immunospots in an integrin-deficient cell line. Confluent monolayers of GD1286 or GD25 cells were infected with hMPV. After 24 h, immunostaining assay was performed, and immunospots formed by hMPV were visualized.

Article Snippet: Twenty picomoles of synthetic small interfering RNAs (siRNAs) targeting either human integrin subtype α5 (sc-29372; Santa Cruz Biotechnology, CA) or αv (sc-29373; Santa Cruz Biotechnology) or control siRNA (sc-37007; Santa Cruz Biotechnology) was transfected into Vero E6 cells (at a confluence of 75%) in 24-well plates using Oligofectamine reagents (Invitrogen) according to the manufacturer's instructions.

Techniques: Infection, Incubation, Binding Assay, Immunostaining

Journal: Journal of Virology

Article Title: Roles of the Putative Integrin-Binding Motif of the Human Metapneumovirus Fusion (F) Protein in Cell-Cell Fusion, Viral Infectivity, and Pathogenesis

doi: 10.1128/JVI.03491-13

Figure Lengend Snippet: Location of the RGD motif in the predicted structure of hMPV F protein. (A) Predicted hMPV F-protein monomer. The structure was predicted using the Modeler (version 9.0) program on the basis of the prefusion crystal structure of RSV F protein (PDB accession no. 4JHW) as the template. The putative integrin-binding sites (R329 and D331) are highlighted. (B) Predicted hMPV F-protein trimer. The surface of each monomer in the F-protein trimer is highlighted in a different color. The RGD motif is located on the contact region of each subunit of the F-protein trimer. (C) Location of the RGD motif in hMPV F-protein monomer. The partial structure of hMPV F protein containing DI, DII, and DIII was solved (PDB accession no. 4DAG) (49). The location of the RGD motif is highlighted. (D) The location of the RGD motif in hMPV F-protein trimer. The surface of each monomer in the F-protein trimer is highlighted in a different color.

Article Snippet: Twenty picomoles of synthetic small interfering RNAs (siRNAs) targeting either human integrin subtype α5 (sc-29372; Santa Cruz Biotechnology, CA) or αv (sc-29373; Santa Cruz Biotechnology) or control siRNA (sc-37007; Santa Cruz Biotechnology) was transfected into Vero E6 cells (at a confluence of 75%) in 24-well plates using Oligofectamine reagents (Invitrogen) according to the manufacturer's instructions.

Techniques: Binding Assay

Journal: BioMed Research International

Article Title: Participation of Integrin α 5 β 1 in the Fibronectin-Mediated Adherence of Enteroaggregative Escherichia coli to Intestinal Cells

doi: 10.1155/2014/781246

Figure Lengend Snippet: Diarrheagenic E. coli bind to integrin α 5 β 1. (a) EAEC, EHEC, and ETEC were added to integrin α 5 β 1- and BSA-coated wells and bacterial binding was detected by ELISA, using anti-O44, anti-O157, and anti-O78 serum, respectively. (b) EAEC strain 042 and 042 aafA were added to integrin α 5 β 1- and BSA-coated wells. EAEC binding was detected by ELISA, using anti-O44 serum. *Significantly different between treatments ( P < 0.05).

Article Snippet: Subconfluent cultures (~40–50%) of HEp-2 cells grown in 6-well plates were transfected with small hairpin RNA (shRNA) for integrin α 5 (sc-29372-SH; Santa Cruz Biotechnology) following the manufacturer's instructions (Santa Cruz Biotechnology).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: BioMed Research International

Article Title: Participation of Integrin α 5 β 1 in the Fibronectin-Mediated Adherence of Enteroaggregative Escherichia coli to Intestinal Cells

doi: 10.1155/2014/781246

Figure Lengend Snippet: Fibronectin/integrin α 5 β 1 complex increases the adhesion of EAEC strain 042. (a) Integrin α 5 β 1- and BSA-coated wells were incubated with increasing concentrations of fibronectin (25, 50, and 100 ng) or (b) with 100 ng of fibronectin (Fn) for 15, 30, or 60 min. Bound fibronectin was detected by ELISA, using anti-fibronectin antibodies. (c) EAEC 042 was added to integrin α 5 β 1- and BSA-coated wells incubated or not with fibronectin. (d) EAEC strain 042 and 042 aafA were added to integrin α 5 β 1- and BSA-coated wells incubated with fibronectin. EAEC binding was detected by ELISA using anti-O44 serum. The bars represent the mean for three experiments, with the error bars indicating standard deviation. *Significantly different between treatments ( P < 0.05).

Article Snippet: Subconfluent cultures (~40–50%) of HEp-2 cells grown in 6-well plates were transfected with small hairpin RNA (shRNA) for integrin α 5 (sc-29372-SH; Santa Cruz Biotechnology) following the manufacturer's instructions (Santa Cruz Biotechnology).

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

Journal: BioMed Research International

Article Title: Participation of Integrin α 5 β 1 in the Fibronectin-Mediated Adherence of Enteroaggregative Escherichia coli to Intestinal Cells

doi: 10.1155/2014/781246

Figure Lengend Snippet: AafA binding to fibronectin/integrin α 5 β 1 complex. (a) AafA- dsc protein was added to integrin α 5 β 1- and BSA-coated wells incubated or not with fibronectin (Fn). AafA- dsc binding was detected by ELISA using anti-AafA antibodies. The bars represent the mean for three experiments, with the error bars indicating standard deviation. *Significantly different between treatments ( P < 0.05). (b) AafA- dsc and fibronectin or (c) AafA- dsc , fibronectin, and integrin α 5 β 1 were mixed and the complex formed was added to control column or a column with anti-AafA antibodies for coimmunoprecipitation analysis. The eluted fraction obtained from control column (E1 and E3) or a column with anti-AafA antibodies (E2 and E4) was analyzed by Dot blot, using anti-AafA, anti-integrin α 5, and anti-fibronectin antibodies. The resulting autoradiography was scanned and signal intensity was quantified by UN-SCAN-IT 6.1 software. One representative experiment of three independent experiments with similar result is shown.

Article Snippet: Subconfluent cultures (~40–50%) of HEp-2 cells grown in 6-well plates were transfected with small hairpin RNA (shRNA) for integrin α 5 (sc-29372-SH; Santa Cruz Biotechnology) following the manufacturer's instructions (Santa Cruz Biotechnology).

Techniques: Binding Assay, Incubation, Enzyme-linked Immunosorbent Assay, Standard Deviation, Control, Dot Blot, Autoradiography, Software

Journal: BioMed Research International

Article Title: Participation of Integrin α 5 β 1 in the Fibronectin-Mediated Adherence of Enteroaggregative Escherichia coli to Intestinal Cells

doi: 10.1155/2014/781246

Figure Lengend Snippet: Adhesion of EAEC strain 042 to T84 cells. T84 cells preincubated with DMEM only (control) or medium supplemented with fibronectin (Fn), Fn and anti-integrin α 5 β 1 (RGD), or anti-integrin α 5 β 1 ( α 5) were infected with EAEC strain 042. The number of adherent bacteria was determined by colony forming unit counts (CFU). *Significantly different between treatments ( P < 0.05).

Article Snippet: Subconfluent cultures (~40–50%) of HEp-2 cells grown in 6-well plates were transfected with small hairpin RNA (shRNA) for integrin α 5 (sc-29372-SH; Santa Cruz Biotechnology) following the manufacturer's instructions (Santa Cruz Biotechnology).

Techniques: Control, Infection, Bacteria

Journal: BioMed Research International

Article Title: Participation of Integrin α 5 β 1 in the Fibronectin-Mediated Adherence of Enteroaggregative Escherichia coli to Intestinal Cells

doi: 10.1155/2014/781246

Figure Lengend Snippet: Integrin α 5 expression knockdown reduces EAEC strain 042 fibronectin-mediated binding to epithelial cells. HEp-2 cells nontransfected and transfected with scrambled or integrin α 5 shRNA were preincubated with DMEM only (control) or medium supplemented with fibronectin (Fn) and then infected with EAEC strain 042. Numbers of adherent bacteria were determined by colony forming unit counts (CFU). *Significantly different compared to control treatment ( P < 0.05).

Article Snippet: Subconfluent cultures (~40–50%) of HEp-2 cells grown in 6-well plates were transfected with small hairpin RNA (shRNA) for integrin α 5 (sc-29372-SH; Santa Cruz Biotechnology) following the manufacturer's instructions (Santa Cruz Biotechnology).

Techniques: Expressing, Knockdown, Binding Assay, Transfection, shRNA, Control, Infection, Bacteria

Journal: Frontiers in Oncology

Article Title: Integrin VLA-5 and FAK are Good Targets to Improve Treatment Response in the Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia

doi: 10.3389/fonc.2014.00112

Figure Lengend Snippet: The expression level of integrin α5 subunit in Ph+ leukemia cell line SUP-B15 is significantly increased after serum starvation . (A) The percentages of cells expressing integrin α4, α5, and β1 subunits before and after serum starvation for 7 h detected by flow cytometry. (B) The mean fluorescent intensity (MFI) of integrin α4, α5, and β1 subunits from whole alive cell population before and after serum starvation for 7 h detected by flow cytometry.

Article Snippet: Human integrin α5 shRNA lentiviral particles (Catalog # sc-29372-V, Santa Cruz) were thawed at room temperature and added to leukemia cell suspension in 15 ml conical tubes and were spun at 800 × g (2500 rpm) for 90 min at 37°C.

Techniques: Expressing, Flow Cytometry

Journal: Frontiers in Oncology

Article Title: Integrin VLA-5 and FAK are Good Targets to Improve Treatment Response in the Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia

doi: 10.3389/fonc.2014.00112

Figure Lengend Snippet: Integrin α5 subunit antibody inhibits the adhesion of Ph+ leukemia cells to human fibronectin and enhances the killing of imatinib . (A) Cell adhesion assay showed that α5 subunit inhibitory antibody (CD49e, clone IIA1 BD Biosciences) was the only tested integrin antibody that significantly inhibited the adhesion of Ph+ leukemia cells to fibronectin with adhesion percentage of 6.6 ± 3.8% compared with control IgG of 44.8 ± 7.9%, p < 0.01. (B) . Annexin-V plus PI by flow cytometry. (i) P2 gate identifies the CD38 positive cell population representing SUP-B15 Ph+ leukemia cells. (ii) Ph+ leukemia cells grown on HS-5 stromal cells. (iii) Ph+ leukemia cells treated with 10 μM imatinib (IM). (iv) SUP-B15 cultured with stromal cells treated with 10 μM imatinib. (v) SUP-B15 cultured with stromal cells treated with 10 μM imatinib and 10 μg/ml anti-α5 Ab. (C) Quantitative analysis for apoptosis rate of different conditions. *After culture on HS-5 cells for 24 h, the apoptosis rate of leukemia cells SUP-B15 decreased from 29.4 ± 2.3 to 16.7 ± 3%, p < 0.05. # When inhibitory antibody to integrin α5 was combined with imatinib to treat Ph+ leukemia cells cultured on stromal cells, the apoptosis rate 38.0 ± 8.0% was significantly increased compared with imatinib by itself 25.7 ± 3.3%, p < 0.05.

Article Snippet: Human integrin α5 shRNA lentiviral particles (Catalog # sc-29372-V, Santa Cruz) were thawed at room temperature and added to leukemia cell suspension in 15 ml conical tubes and were spun at 800 × g (2500 rpm) for 90 min at 37°C.

Techniques: Cell Adhesion Assay, Control, Flow Cytometry, Cell Culture

Journal: Frontiers in Oncology

Article Title: Integrin VLA-5 and FAK are Good Targets to Improve Treatment Response in the Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia

doi: 10.3389/fonc.2014.00112

Figure Lengend Snippet: Blocking integrin α5 affects the engraftment of Ph+ leukemia cells in immunodeficient mice . (A) The incubation for 1 h of Ph+ leukemia cells with disintegrin, a peptide inhibitor of integrins, impaired the engraftment of leukemia in NSG mice. Representative figures of n = 2 showed bioluminescence imaging from day 7 to 28 after inoculation of leukemia cells. (B) Animal total body bioluminescence was measured using the Xenogen IVIS Imaging System 200 Series with total imaging time of 2 min and compared with control animals that received cells that were not treated with Echistatin. (C) Anti-integrin α5 inhibitory antibodies clone IIA1 (BD Biosciences) and clone P1D6 (Millipore) decreased the engraftment of Ph+ leukemia cells in the bone marrow of NOD/SCID mice ( n = 2).

Article Snippet: Human integrin α5 shRNA lentiviral particles (Catalog # sc-29372-V, Santa Cruz) were thawed at room temperature and added to leukemia cell suspension in 15 ml conical tubes and were spun at 800 × g (2500 rpm) for 90 min at 37°C.

Techniques: Blocking Assay, Incubation, Imaging, Control

Journal: Frontiers in Oncology

Article Title: Integrin VLA-5 and FAK are Good Targets to Improve Treatment Response in the Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia

doi: 10.3389/fonc.2014.00112

Figure Lengend Snippet: Knocking down integrin α5 delayed the engraftment of Ph+ leukemia cells in immunodeficient NOD/SCID mice . (A) Real-time QPCR assay showed that we had successfully knocked down the expression level of the integrin α5 knocking in clone 10. The cycle threshold (Ct) of the target gene (integrin α5) was normalized to the chosen reference gene GAPHD. Relative quantification, R = 2 −(ΔCt sample − ΔCt control) . Result shows the fold change normalized to SUP-LUC2 cells. (B) Western blot showed a reduced protein expression level of integrin α5 in clone 10. (C) Flow cytometry showed reduced mean fluorescent intensity of integrin α5 in clone 10. (D) Representative figures showed bioluminescent imaging at 2 weeks, and 5–8 weeks post leukemia cells inoculation. (E) Animal total body bioluminescence was quantified to compare with control. **The levels of bioluminescence became most significantly different 2 months post inoculation between the α5 knock-down group and control group [mean (SD) radiance vs. control, 5.3 (0.1) vs. 2.1 (0.2) × 10 6 p/s/cm 2 /sr, p < 0.01 at 2 months, n = 3.].

Article Snippet: Human integrin α5 shRNA lentiviral particles (Catalog # sc-29372-V, Santa Cruz) were thawed at room temperature and added to leukemia cell suspension in 15 ml conical tubes and were spun at 800 × g (2500 rpm) for 90 min at 37°C.

Techniques: Expressing, Quantitative Proteomics, Control, Western Blot, Flow Cytometry, Imaging, Knockdown