Journal: The Journal of Experimental Medicine

Article Title: Sialyltransferase ST3Gal-IV controls CXCR2-mediated firm leukocyte arrest during inflammation

doi: 10.1084/jem.20070846

Figure Lengend Snippet: Leukocyte adhesion during inflammation in vivo. (A) Number of adherent leukocytes (mean ± SEM per mm 2 of surface area) in unstimulated mouse cremaster muscle venules of WT control and ST3Gal-IV −/− mice. (B) Increase in the number of adherent leukocytes (mean ± SEM per mm 2 of surface area) 3 min after systemic injection of 600 ng CXCL1 or CXCL8 in WT control, ST3Gal-IV −/− , WT control (plus 4 μg PTx), ST3Gal-IV −/− (plus 4 μg PTx), and CXCR2 bm−/− mice. PTx was injected 3 h before cremaster muscle exteriorization. (C) Leukocyte adhesion (mean ± SEM) was observed in 3-h TNF-α–treated cremaster muscle venules of WT control, ST3Gal-IV −/− , and CXCR2 bm−/− mice. Additionally, pretreatment of TNF-α–treated WT control, ST3Gal-IV −/− , and CXCR2 bm−/− mice was conducted with 4 μg PTx and/or 100 μg E-selectin mAb 9A9 5 min before TNF-α injection. Data in A–C were obtained from at least three independent experiments per group. *, P < 0.05 versus WT control mice (A and C); *, P < 0.05 versus WT control mice treated with CXCL1 or CXCL8, respectively (B). Videos 1 and 2 are available at http://www.jem.org/cgi/content/full/jem.20070846/DC1 .

Article Snippet: E-selectin blocking mAb 9A9 (rat IgG1; 30 μg per mouse) was a gift from B. Wolitzky (MitoKor, San Diego, CA).

Techniques: In Vivo, Control, Injection

Journal: The Journal of Experimental Medicine

Article Title: Sialyltransferase ST3Gal-IV controls CXCR2-mediated firm leukocyte arrest during inflammation

doi: 10.1084/jem.20070846

Figure Lengend Snippet: Cremaster muscle whole mount. Giemsa-stained whole-mounts of TNF-α–treated cremaster muscles of WT, ST3Gal-IV −/− , and CXCR2 bm−/− mice were analyzed for the number of intravascular (A) and perivascular (B) leukocytes (mean ± SEM per mm 2 surface area). In addition, two typical micrographs are presented illustrating intra- and perivascular leukocyte distribution in TNF-α–treated cremaster muscle whole mounts of WT control mice (C) and ST3Gal-IV −/− mice pretreated with E-selectin blocking mAb 9A9 (D). Data in A and B were obtained from at least three independent experiments per group. *, P < 0.05 versus WT mice. Bars, 50 μm.

Article Snippet: E-selectin blocking mAb 9A9 (rat IgG1; 30 μg per mouse) was a gift from B. Wolitzky (MitoKor, San Diego, CA).

Techniques: Staining, Muscles, Control, Blocking Assay

Journal: The Journal of Experimental Medicine

Article Title: Sialyltransferase ST3Gal-IV controls CXCR2-mediated firm leukocyte arrest during inflammation

doi: 10.1084/jem.20070846

Figure Lengend Snippet: Adhesion in the flow chamber and transmigration of ST3Gal-IV −/− neutrophils in vitro. The number of rolling and adherent leukocytes from ST3Gal-IV −/− and WT mice was determined in blood-perfused microflow chambers. (A) Rolling and (B) adherent leukocytes in autoperfused flow chambers (5.94 dynes/cm 2 ) coated with 20 μg/ml P-selectin and 15 μg/ml ICAM-1 with or without 15 μg/ml CXCL1. Data in A and B are presented as mean ± SEM from at least four mice and four flow chambers. *, P < 0.05 versus WT mice (B). (C) The percentage of transmigrated neutrophils from WT control mice, ST3Gal-IV −/− mice, and WT control mice pretreated with neuraminidase was assessed in a transwell assay in which transwells were coated with an immortalized endothelial cell line, b.End5. In addition, transmigration of WT control neutrophils through a neuraminidase-pretreated b.End5 monolayer was analyzed. Data in C were determined from at least three independent experiments per group. *, P < 0.05 versus untreated WT control neutrophils (C). FOV, field of view.

Article Snippet: E-selectin blocking mAb 9A9 (rat IgG1; 30 μg per mouse) was a gift from B. Wolitzky (MitoKor, San Diego, CA).

Techniques: Transmigration Assay, In Vitro, Control, Transwell Assay

Journal: Journal of Controlled Release

Article Title: Targeting adenovirus gene delivery to activated tumour-associated vasculature via endothelial selectins

doi: 10.1016/j.jconrel.2010.10.011

Figure Lengend Snippet: Streptococcal protein G as a platform system for targeting E-selectin in vitro . (a) Diagram illustrating concept of retargeting pcAdluc by linking antibodies via protein G. Polymer coated virus shown in grey. (b) Retargeting protein G-pHPMA-coated adenovirus via E-selectin in rhTNF-α-activated HUVECs. Ad, unmodified adenovirus; StrepGpcAd, protein G-pHPMA-adenovirus; IgG-StrepGpcAd, isotype control-protein G-pHPMA-adenovirus; and MHES-StrepGpcAd, MHES-retargeted-protein G-pHPMA-adenovirus. Results are representative of three different experiments. Error bars are ± standard deviation. P values calculated using a one-way ANOVA (analysis of variance). *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: RMES (mouse IgG2a anti-rat E-selectin) monoclonal antibody was purchased from BD Pharmingen (Oxford, UK).

Techniques: In Vitro, Standard Deviation

Journal: Journal of Controlled Release

Article Title: Targeting adenovirus gene delivery to activated tumour-associated vasculature via endothelial selectins

doi: 10.1016/j.jconrel.2010.10.011

Figure Lengend Snippet: Retargeting protein G-pHPMA-coated adenovirus using alternative Fc-bearing ligands targeting murine endothelia. (a) Transduction of murine bEnd3 cells using StrepGpcAdluc retargeted via E-selectin with a rat anti-mouse E-selectin IgG2a monoclonal antibody (RMES mAb). (b) Transduction of murine bEnd3 cells using StrepGpcAdluc retargeted via P-selectin with a chimeric P-selectin Glycoprotein Ligand-1 (PSGL-1)-Fc fusion protein. In each case prior to infection cells were treated with recombinant murine-TNF-α (rmTNF-α, 120 ng/ml) for 5 h at 37 °C. Ad, unmodified adenovirus; pcAd, pHPMA-adenovirus; StrepGpcAd, protein G-pHPMA-adenovirus; RMES-StrepGpcAd, RMES retargeted-protein G-pHPMA-adenovirus; and PSGL-1-Fc-StrepGpcAd, PSGL-1 retargeted-protein G-pHPMA-adenovirus. Results are representative of at least three different experiments. Error bars are ± standard deviation. P values were calculated using a one-way ANOVA (analysis of variance). *P < 0.05, **P < 0.01.

Article Snippet: RMES (mouse IgG2a anti-rat E-selectin) monoclonal antibody was purchased from BD Pharmingen (Oxford, UK).

Techniques: Transduction, Infection, Recombinant, Standard Deviation

Journal: Journal of Controlled Release

Article Title: Targeting adenovirus gene delivery to activated tumour-associated vasculature via endothelial selectins

doi: 10.1016/j.jconrel.2010.10.011

Figure Lengend Snippet: Pharmacokinetics of PSGL-1-Fc retargeted virus. (a) Blood circulation profile of PSGL-1-Fc-StrepGpcAd (squares) and Adluc (diamonds) in vivo following intravenous administration of 1e10 virus particles. (b) Number of virus genomic copies recovered from HepG2 xenograft tumours following administration of Adluc, StrepGpcAdluc, IgG1-StrepGpcAdluc or PSGL-1-Fc-StrepGpcAdluc. P values were calculated using a one-way ANOVA (analysis of variance). *P < 0.05.

Article Snippet: RMES (mouse IgG2a anti-rat E-selectin) monoclonal antibody was purchased from BD Pharmingen (Oxford, UK).

Techniques: In Vivo

Journal: PLoS ONE

Article Title: CD44v4 Is a Major E-Selectin Ligand that Mediates Breast Cancer Cell Transendothelial Migration

doi: 10.1371/journal.pone.0001826

Figure Lengend Snippet: A: differential migration of breast cancer cells across HUVEC monolayers pre-activated with TNF-α. B: migration of MDA-MB-231 across HUVEC monolayers in the presence of anti-E-selectin, anti-ICAM-1, and anti-VLA-4 antibodies (25 µg/ml each), respectively. HUVEC monolayers were pre-incubated with endothelial medium containing 25 ng/ml TNF-α for 6 h, and migration was terminated after overnight incubation at 37°C. All data are expressed as the mean±SD ( n = 3) of a typical experiment and similar results were obtained in at least three separate experiments.

Article Snippet: The monoclonal antibodies used in the experiments and their resources: mouse anti-human E-selectin mAb (MAB575), mouse anti-human CD44H (clone 2C5), mouse anti-human CD44v3 (clone 3G5), mouse anti-human CD44v6 (clone 2F10) and mouse anti-human CD44v4/5 (clone 3D2) (R&D Systems, Minneapolis, MN); Anti-CD44v4 mAb (Lab Vision, Fremont, CA); Anti-human LFA-1 and anti-human VLA-4 mAbs (BD Pharmingen, San Diego, CA); Anti-sLe x mAb (Clone KM93) (Chemicon, Temecula, CA); HRP-conjugated goat anti-mouse and anti-rabbit IgG (Jackson ImmunoResearch Lab, West Grove, PA); Alexa Fluor 488(495/519) and Alexa Fluor 568(578/603)-conjugated secondary antibodies (Molecular Probes, Eugene, OR).

Techniques: Migration, Incubation

Journal: PLoS ONE

Article Title: CD44v4 Is a Major E-Selectin Ligand that Mediates Breast Cancer Cell Transendothelial Migration

doi: 10.1371/journal.pone.0001826

Figure Lengend Snippet: Trypsin-elicited breast cancer cells were surface labeled with soluble exE-selectin/Fc or Fc-only as detailed in the . The bound Fc recombinants were detected by Alexa Fluor 488-conjugated goat anti-rabbit Fc and measured by FACS. In separated experiments, tumor cells were treated with neuraminidase to remove surface sLe x moieties before exE-selectin/Fc binding.

Article Snippet: The monoclonal antibodies used in the experiments and their resources: mouse anti-human E-selectin mAb (MAB575), mouse anti-human CD44H (clone 2C5), mouse anti-human CD44v3 (clone 3G5), mouse anti-human CD44v6 (clone 2F10) and mouse anti-human CD44v4/5 (clone 3D2) (R&D Systems, Minneapolis, MN); Anti-CD44v4 mAb (Lab Vision, Fremont, CA); Anti-human LFA-1 and anti-human VLA-4 mAbs (BD Pharmingen, San Diego, CA); Anti-sLe x mAb (Clone KM93) (Chemicon, Temecula, CA); HRP-conjugated goat anti-mouse and anti-rabbit IgG (Jackson ImmunoResearch Lab, West Grove, PA); Alexa Fluor 488(495/519) and Alexa Fluor 568(578/603)-conjugated secondary antibodies (Molecular Probes, Eugene, OR).

Techniques: Labeling, Binding Assay

Journal: PLoS ONE

Article Title: CD44v4 Is a Major E-Selectin Ligand that Mediates Breast Cancer Cell Transendothelial Migration

doi: 10.1371/journal.pone.0001826

Figure Lengend Snippet: A: After blocking with 5% normal goat serum in PBS, breast cancer cells cultured in a tissue culture dish were incubated with exE-selectin/Fc (10 µg/ml) in blocking solution for 1 h at 4°C. Following three washes, cells were fixed with 3.5% paraformaldehyde (5 min, 20°C) and the bound exE-selectin/Fc was detected by Alexa Fluor 488-conjugated anti-rabbit Fc. Cell surface labeling with Fc-only served as a background control. B: To determine the localization of E-selectin ligand on metastatic tumor cell surfaces during migration, MDA-MB-231 cells were double labeled with soluble exE-selectin/Fc for E-selectin ligand (left panel) and rhodamine-conjugated phalloidin for F-actin (right panel). Bar, 20 µm.

Article Snippet: The monoclonal antibodies used in the experiments and their resources: mouse anti-human E-selectin mAb (MAB575), mouse anti-human CD44H (clone 2C5), mouse anti-human CD44v3 (clone 3G5), mouse anti-human CD44v6 (clone 2F10) and mouse anti-human CD44v4/5 (clone 3D2) (R&D Systems, Minneapolis, MN); Anti-CD44v4 mAb (Lab Vision, Fremont, CA); Anti-human LFA-1 and anti-human VLA-4 mAbs (BD Pharmingen, San Diego, CA); Anti-sLe x mAb (Clone KM93) (Chemicon, Temecula, CA); HRP-conjugated goat anti-mouse and anti-rabbit IgG (Jackson ImmunoResearch Lab, West Grove, PA); Alexa Fluor 488(495/519) and Alexa Fluor 568(578/603)-conjugated secondary antibodies (Molecular Probes, Eugene, OR).

Techniques: Blocking Assay, Cell Culture, Incubation, Labeling, Control, Migration

Journal: PLoS ONE

Article Title: CD44v4 Is a Major E-Selectin Ligand that Mediates Breast Cancer Cell Transendothelial Migration

doi: 10.1371/journal.pone.0001826

Figure Lengend Snippet: A: Gel-code staining of proteins eluted from an exE-selectin/Fc affinity column. A 170 kD protein indicated by the arrow was selected as a potential ligand of E-selectin in tumor cells since 130 kD and 50 kD protein bands were confirmed as exE-selectin/Fc and IgG heavy chain, respectively. B: matching of the 10 amino acid N-terminal peptide sequence (red) of the 170 kD purified protein with that of human CD44. C: Western blot analysis of five breast cancer cell lines with anti-CD44H antibody.

Article Snippet: The monoclonal antibodies used in the experiments and their resources: mouse anti-human E-selectin mAb (MAB575), mouse anti-human CD44H (clone 2C5), mouse anti-human CD44v3 (clone 3G5), mouse anti-human CD44v6 (clone 2F10) and mouse anti-human CD44v4/5 (clone 3D2) (R&D Systems, Minneapolis, MN); Anti-CD44v4 mAb (Lab Vision, Fremont, CA); Anti-human LFA-1 and anti-human VLA-4 mAbs (BD Pharmingen, San Diego, CA); Anti-sLe x mAb (Clone KM93) (Chemicon, Temecula, CA); HRP-conjugated goat anti-mouse and anti-rabbit IgG (Jackson ImmunoResearch Lab, West Grove, PA); Alexa Fluor 488(495/519) and Alexa Fluor 568(578/603)-conjugated secondary antibodies (Molecular Probes, Eugene, OR).

Techniques: Staining, Affinity Column, Sequencing, Purification, Western Blot

Journal: PLoS ONE

Article Title: CD44v4 Is a Major E-Selectin Ligand that Mediates Breast Cancer Cell Transendothelial Migration

doi: 10.1371/journal.pone.0001826

Figure Lengend Snippet: A: RT-PCR amplification and DNA sequence of CD44 extracellular domain from various breast cancer cells and colonic HT29 cells. B: Western blot analysis of the 170 kD purified protein with antibodies specific to sLe x moieties and different CD44 variant isoforms, respectively. C: Specific binding of the 170 kD purified protein (CD44v4) to E-selectin through sLe x moieties. The data are represented as the mean±SD of three different experiments.

Article Snippet: The monoclonal antibodies used in the experiments and their resources: mouse anti-human E-selectin mAb (MAB575), mouse anti-human CD44H (clone 2C5), mouse anti-human CD44v3 (clone 3G5), mouse anti-human CD44v6 (clone 2F10) and mouse anti-human CD44v4/5 (clone 3D2) (R&D Systems, Minneapolis, MN); Anti-CD44v4 mAb (Lab Vision, Fremont, CA); Anti-human LFA-1 and anti-human VLA-4 mAbs (BD Pharmingen, San Diego, CA); Anti-sLe x mAb (Clone KM93) (Chemicon, Temecula, CA); HRP-conjugated goat anti-mouse and anti-rabbit IgG (Jackson ImmunoResearch Lab, West Grove, PA); Alexa Fluor 488(495/519) and Alexa Fluor 568(578/603)-conjugated secondary antibodies (Molecular Probes, Eugene, OR).

Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Sequencing, Western Blot, Purification, Variant Assay, Binding Assay

Journal: PLoS ONE

Article Title: CD44v4 Is a Major E-Selectin Ligand that Mediates Breast Cancer Cell Transendothelial Migration

doi: 10.1371/journal.pone.0001826

Figure Lengend Snippet: A and B: siRNA transfection-induced CD44v4 downregulation in MDA-MB-231 cells as shown by Western blot (A) and immunofluorescence labeling (B), respectively. C and D: reduction of MDA-MB-231 cell adhesion to immobilized recombinant exE-selectin/Fc (C) and TNF-α pre-activated HUVEC monolayers (D) after CD44v4 downregulation via siRNA. E: reduction of MDA-MB-231 cell transendothelial migration by CD44v4 downregulation. The data are representative of three independent experiments.

Article Snippet: The monoclonal antibodies used in the experiments and their resources: mouse anti-human E-selectin mAb (MAB575), mouse anti-human CD44H (clone 2C5), mouse anti-human CD44v3 (clone 3G5), mouse anti-human CD44v6 (clone 2F10) and mouse anti-human CD44v4/5 (clone 3D2) (R&D Systems, Minneapolis, MN); Anti-CD44v4 mAb (Lab Vision, Fremont, CA); Anti-human LFA-1 and anti-human VLA-4 mAbs (BD Pharmingen, San Diego, CA); Anti-sLe x mAb (Clone KM93) (Chemicon, Temecula, CA); HRP-conjugated goat anti-mouse and anti-rabbit IgG (Jackson ImmunoResearch Lab, West Grove, PA); Alexa Fluor 488(495/519) and Alexa Fluor 568(578/603)-conjugated secondary antibodies (Molecular Probes, Eugene, OR).

Techniques: Transfection, Western Blot, Immunofluorescence, Labeling, Recombinant, Migration