Journal: Oncotarget

Article Title: CXCR6-CXCL16 axis promotes prostate cancer by mediating cytoskeleton rearrangement via Ezrin activation and α v β 3 integrin clustering

doi: 10.18632/oncotarget.6944

Figure Lengend Snippet: Panel-A. Expression of integrin α v β 3 in PCa cell lines. Untreated or CXCL16 treated PCa cells stained for integrin α v β 3 and nucleus stained with Draq5. Panel-B. Quantitative analysis of CXCL16 induced integrin α v β 3 clustering using ImageStream100 image based flow cytometer. Bright field (white), Integrin α v β 3 (green) and Draq5 (red), composite images for representative PC3 and LNCaP cells are shown. Panel-C. Percentage of cells showing α v β 3 integrin clustering in response to CXCL16 treatment with or without PKC/FAK inhibition. Integrin clustering was quantified using two standard image-based software analysis features: Area aspect ratio and Radial delta centroid, which demonstrated three types of cell population in samples: Uniform (global distribution of integrin α v β 3 on cell surface; cells with high aspect ratio with low radial delta centroid values), Capped (integrin α v β 3 clustered in a specific cell area; cells with high aspect ratio values and relatively low radial delta centroid) and Atypical (cell debris; cells with smaller aspect ratio and low radial delta centroid).

Article Snippet: Briefly, the cells were stained with 1 μg of FITC-conjugated mouse anti-human α v β 3 antibody (BD PharMingen) at 4°C for 30 min.

Techniques: Expressing, Staining, Flow Cytometry, Inhibition, Software

Journal: PLoS Pathogens

Article Title: CD36 Recruits α 5 β 1 Integrin to Promote Cytoadherence of P. falciparum -Infected Erythrocytes

doi: 10.1371/journal.ppat.1003590

Figure Lengend Snippet: (A) Adhesion of IRBC to endothelial monolayers pre-incubated with 20 µM RAD or 5–50 µM RGD peptide for 30 min at 37°C in 5% CO 2 . A 1% hematoctit suspension of IRBC from the lab-adapted parasite clone 7G8 was drawn over the monolayers at 1 dyne/cm 2 . Results shown are the mean number of adherent IRBC/mm 2 in 4–6 microscopic fields (20×) after 7 min of infusion (n = 6). (B) Adhesion of 7G8 parasites to endothelial monolayers pre-incubated with 20 µM cRADfV or cRGDfV peptide (n = 3). (C) Adhesion of 3 clinical parasite isolates to endothelial monolayers pre-incubated with 20 µM RAD or RGD peptide (n = 10 for 3 clinical isolates each tested in 3 to 4 independent experiments). (D) Adhesion of clinical isolates to endothelial monolayers pre-incubated with control IgG 1 , an inhibitory anti-β 1 integrin mAb TDM29 or the activating anti-β 1 integrin mAb TS2/16 at 10 mg/ml (n = 3 for 3 clinical isolates each tested in 1 independent experiment). (E) Adhesion of clinical isolates to endothelial monolayers pre-incubated with control IgG 1 , and an inhibitory anti-α 5 integrin mAb JBS5 at 10 µg/ml (n = 3 for 3 clinical isolates each tested in 1 independent experiment). (F) Adhesion of clinical isolates to endothelial monolayers pre-incubated with control IgG 1 , and an inhibitory anti-α v β 3 integrin mAb 23C6 at 10 µg/ml (n = 3 for 3 clinical isolates each tested in 1 independent experiment).

Article Snippet: The following mAb were used: anti-human CD36 clone FA6-152 (Beckman Coulter Canada, Inc., Mississauga, ON); anti-human integrin β 1 clones TDM 29 and TS2/16 (Millipore); FITC- and PE-labelled anti-human integrin β 1 clone MEM-101A (Abcam, Cambridge, MA); anti-human α 5 clones JBS5 (Millipore); anti-human ICAM-1 clone 84H10 (R&D Systems, Inc Minneapolis, MN); anti-human α v β 3 clone 23C6 (Chemicon International); mouse IgG1 clone 11711 (R&D Systems); anti-phospho-Tyr418Src (BioSource; Invitrogen), anti-His-tag (His-probe (H-15)) (Santa Cruz Biotechnology Inc., Santa Cruz, CA); FITC goat anti-mouse IgG 1 (Becton Dickinson, San Diego, CA); and Alexa Fluor 488 or 568 goat anti-mouse IgG 1 antibodies and rhodamine-phalloidin (Molecular Probes, Invitrogen).

Techniques: Incubation

Journal: PLoS Pathogens

Article Title: CD36 Recruits α 5 β 1 Integrin to Promote Cytoadherence of P. falciparum -Infected Erythrocytes

doi: 10.1371/journal.ppat.1003590

Figure Lengend Snippet: (A) Schematic representation of a typical AFM force curve which depicts a) approach of the IRBC to an endothelial monolayer and b) retraction of the IRBC. The bar indicates the force of detachment that is used as a measure of adhesive strength between IRBC and endothelium. (B) Force measurement on endothelial monolayers pre-incubated with 20 µM RAD or RGD peptide for 30 min at 37°C in 5% CO 2 (n = 7). (C) Force measurement on endothelial monolayers pre-incubated with control IgG 1 , an inhibitory anti-β 1 integrin mAb TDM29 or the activating anti-β 1 integrin mAb TS2/16 at 10 µg/ml (n = 4). (D) Force measurement on endothelial monolayers pre-incubated with 20 µM cRADfV or cRGDfV peptide (n = 3). For each experiment, 2 IRBC were brought into contact with 3 HDMEC. Contact for 5 min was maintained with a constant force of 150 pN.

Article Snippet: The following mAb were used: anti-human CD36 clone FA6-152 (Beckman Coulter Canada, Inc., Mississauga, ON); anti-human integrin β 1 clones TDM 29 and TS2/16 (Millipore); FITC- and PE-labelled anti-human integrin β 1 clone MEM-101A (Abcam, Cambridge, MA); anti-human α 5 clones JBS5 (Millipore); anti-human ICAM-1 clone 84H10 (R&D Systems, Inc Minneapolis, MN); anti-human α v β 3 clone 23C6 (Chemicon International); mouse IgG1 clone 11711 (R&D Systems); anti-phospho-Tyr418Src (BioSource; Invitrogen), anti-His-tag (His-probe (H-15)) (Santa Cruz Biotechnology Inc., Santa Cruz, CA); FITC goat anti-mouse IgG 1 (Becton Dickinson, San Diego, CA); and Alexa Fluor 488 or 568 goat anti-mouse IgG 1 antibodies and rhodamine-phalloidin (Molecular Probes, Invitrogen).

Techniques: Incubation

Journal: PLoS Pathogens

Article Title: CD36 Recruits α 5 β 1 Integrin to Promote Cytoadherence of P. falciparum -Infected Erythrocytes

doi: 10.1371/journal.ppat.1003590

Figure Lengend Snippet: HDMEC were grown to 95% confluence in ibidi VI chambers. (A) IRBC purified on a MACS separation column were added to a monolayer at 0.1% hematocrit. An FITC-labelled anti-b1integrin mAb was added at 10 µg/ml. The IRBC interaction with HDMEC was imaged in a humidified chamber with 5% CO 2 at 37°C. Results are representative of 2 experiments. (B) 3D reconstruction of the cup-shaped structure formed by clustered β 1 integrin on the endothelial cell membrane. (C and D) HDMEC monolayers in ibidi VI chambers were incubated with beads coated with IgG 1 , anti-CD36, anti-HIS or PpMC-179, the CD36 binding peptide of PfEMP1, for 30 min at 37°C in 5% CO 2 . The monolayers were washed 2× with HBSS to remove unbound beads prior to fixing with 1% PFA for 30 minutes at room temperature. Fixed cells were stained with an anti-β 1 mAb at 10 µg/ml followed by Alexa 488-labelled anti-mouse IgG 1 (C) or with a PE-labelled anti-β 1 integrin mAb at 10 mg/ml (D). Results shown are representative of 3 experiments. (E) HDMEC monolayers in ibidi chambers transduced with GFP-ICAM-1 were incubated with beads coated with IgG 1 , anti-ICAM-1 and anti-CD36, and processed as in (C) and (D). (F) Unstimulated and TNF-α-stimulated HDMEC monolayers in ibidi chambers were incubated with beads coated with IgG 1 , anti-ICAM-1 and anti-CD36, and processed as in (C) and (D). The actin cytoskeleton was visualized by permeabilizing the cells for 5 min with 0.2% TX-100 prior to adding 1 µl of rhodamine-phalloidin in 60 µl HBSS to each chamber for 30 min at room temperature. Results shown are for anti-ICAM-1 coated beads only, and are representative of 3 experiments. All images were taken on an Olympus IX81 inverted confocal microscope (Center Valley, Pa) with Fluorview 1000 acquisition software using a PlanAPO 60× N.A. 1.42 oil immersion objective. White arrows indicate representative sites of β 1 integrin or ICAM-1 recruitment. (G) Quantification of microscopic changes seen in (F). For every condition in every experiment, three randomly selected microscopic fields at 60× magnification were selected. Each field with 15–25 adherent beads were scanned, and adherent beads associated with protein recruitment were scored as positive. Beads partially in the field of view or rings with no adherent beads were excluded in the enumeration. Results are expressed as positive adherent beads/total adherent beads ×100% (n = 3).

Article Snippet: The following mAb were used: anti-human CD36 clone FA6-152 (Beckman Coulter Canada, Inc., Mississauga, ON); anti-human integrin β 1 clones TDM 29 and TS2/16 (Millipore); FITC- and PE-labelled anti-human integrin β 1 clone MEM-101A (Abcam, Cambridge, MA); anti-human α 5 clones JBS5 (Millipore); anti-human ICAM-1 clone 84H10 (R&D Systems, Inc Minneapolis, MN); anti-human α v β 3 clone 23C6 (Chemicon International); mouse IgG1 clone 11711 (R&D Systems); anti-phospho-Tyr418Src (BioSource; Invitrogen), anti-His-tag (His-probe (H-15)) (Santa Cruz Biotechnology Inc., Santa Cruz, CA); FITC goat anti-mouse IgG 1 (Becton Dickinson, San Diego, CA); and Alexa Fluor 488 or 568 goat anti-mouse IgG 1 antibodies and rhodamine-phalloidin (Molecular Probes, Invitrogen).

Techniques: Purification, Incubation, Binding Assay, Staining, Transduction, Microscopy, Software

Journal: PLoS Pathogens

Article Title: CD36 Recruits α 5 β 1 Integrin to Promote Cytoadherence of P. falciparum -Infected Erythrocytes

doi: 10.1371/journal.ppat.1003590

Figure Lengend Snippet: HMEC-1 monolayers in ibidi chambers transduced with GFP-CD36 were incubated with beads coated with IgG 1 or anti-CD36 for 30 min at 37°C in 5% CO 2 . The slides were fixed and stained as described in using a PE-labelled anti-β 1 integrin mAb. Results shown are representative of 3 independent experiments.

Article Snippet: The following mAb were used: anti-human CD36 clone FA6-152 (Beckman Coulter Canada, Inc., Mississauga, ON); anti-human integrin β 1 clones TDM 29 and TS2/16 (Millipore); FITC- and PE-labelled anti-human integrin β 1 clone MEM-101A (Abcam, Cambridge, MA); anti-human α 5 clones JBS5 (Millipore); anti-human ICAM-1 clone 84H10 (R&D Systems, Inc Minneapolis, MN); anti-human α v β 3 clone 23C6 (Chemicon International); mouse IgG1 clone 11711 (R&D Systems); anti-phospho-Tyr418Src (BioSource; Invitrogen), anti-His-tag (His-probe (H-15)) (Santa Cruz Biotechnology Inc., Santa Cruz, CA); FITC goat anti-mouse IgG 1 (Becton Dickinson, San Diego, CA); and Alexa Fluor 488 or 568 goat anti-mouse IgG 1 antibodies and rhodamine-phalloidin (Molecular Probes, Invitrogen).

Techniques: Transduction, Incubation, Staining

Journal: PLoS Pathogens

Article Title: CD36 Recruits α 5 β 1 Integrin to Promote Cytoadherence of P. falciparum -Infected Erythrocytes

doi: 10.1371/journal.ppat.1003590

Figure Lengend Snippet: (A) HDMEC monolayers were pre-incubated with PP3 or PP1 at a concentration of 10 µM for 2 hours, or DMSO or BAPTA for 30 min followed by HBSS for 30 min, at 37°C in 5% CO 2 . Anti-CD36 coated beads were then added to the monolayers for 30 min. After unattached beads were washed off, the monolayers were fixed and stained with Alexa 488-labeled anti-β 1 integrin for 1 hr at room temperature. The images were taken as in . Results shown are representative of 3 experiments. (B) Quantification of microscopic changes as described in . (C) HDMEC transduced with GFP-CD36 were either untreated, or pre-incubated with 20 µM RAD/RGD, or IgG 1 /inhibitory anti-β 1 integrin mAb (clone TDM29) at 10 µg/ml for 30 min at 37°C in 5% CO 2 . After the addition of anti-CD36 coated beads for 30 min, the monolayers were fixed and stained as in (A). Quantification of 2 experiments is shown. (D) Anti-CD36 coated beads were added to peptide or antibody-treated HDMEC monolayers as in (C). The monolayers were fixed, permeabilized with 0.2%TX-100 for 5 min, and blocked with 1%BSA+0.003%TX-100 for 30 min. Monolayers were stained with a polyclonal anti-phospho-Src antibody overnight at 4°C followed by goat-anti-rabbit IgG-Alexa 488 for 1 hr at room temperature. The actin cytoskeleton was visualized by adding 1 µl of rhodamine-phalloidin in 60 µl HBSS to each chamber for 30 min at room temperature. Quantification of 2 experiments is shown.

Article Snippet: The following mAb were used: anti-human CD36 clone FA6-152 (Beckman Coulter Canada, Inc., Mississauga, ON); anti-human integrin β 1 clones TDM 29 and TS2/16 (Millipore); FITC- and PE-labelled anti-human integrin β 1 clone MEM-101A (Abcam, Cambridge, MA); anti-human α 5 clones JBS5 (Millipore); anti-human ICAM-1 clone 84H10 (R&D Systems, Inc Minneapolis, MN); anti-human α v β 3 clone 23C6 (Chemicon International); mouse IgG1 clone 11711 (R&D Systems); anti-phospho-Tyr418Src (BioSource; Invitrogen), anti-His-tag (His-probe (H-15)) (Santa Cruz Biotechnology Inc., Santa Cruz, CA); FITC goat anti-mouse IgG 1 (Becton Dickinson, San Diego, CA); and Alexa Fluor 488 or 568 goat anti-mouse IgG 1 antibodies and rhodamine-phalloidin (Molecular Probes, Invitrogen).

Techniques: Incubation, Concentration Assay, Staining, Labeling, Transduction