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: Scientific Reports

Article Title: C 6 -ceramide nanoliposome suppresses tumor metastasis by eliciting PI3K and PKCζ tumor-suppressive activities and regulating integrin affinity modulation

doi: 10.1038/srep09275

Figure Lengend Snippet: Flow cytometry analysis of adhesion molecule expression on MDA-MB-231 (1 st row) and Lu1205 (2 nd row)

Article Snippet: Mouse IgG anti-human α v β 3 (anti-CD51/61, clone 23C6), CD44H, VLA-4 and ICAM-1 (clone BBIG-I1) were purchased from R&D Systems (Minneapolis, MN).

Techniques: Flow Cytometry, Expressing, Control

Journal: Scientific Reports

Article Title: C 6 -ceramide nanoliposome suppresses tumor metastasis by eliciting PI3K and PKCζ tumor-suppressive activities and regulating integrin affinity modulation

doi: 10.1038/srep09275

Figure Lengend Snippet: (a) PKCζ DNbut not PKCα DN and PKCε DN conferred NaL-C 6 -treated cells shear-resistant adhesive capacity. Fibrinogen was coated as substrate, before the parallel plate flow chamber was assembled. Transfected MDA-MB-231 cells were settled onto fibrinogen for 7 min prior to initiation of the assays. Step-load shears (0, 50, 100, 200, 400, 800, and 1600 sec -1 ) were applied to attached cells. The percentage of cells remaining bound to fibrinogen after each shear step was determined (expressed as % cells remaining bound). Results were expressed as mean ± SEM from three independent experiments. * p < 0.05 compared with NaL-C 6 +vector at each shear rate. (b–c)Silencing integrin α v β 3 by siRNA compromised the ability of PKCζ DN (b) or PI3K inhibitors (c) to rescue cell adhesion. Scrambled or integrin α v β 3 -targeting siRNA was introduced into MDA-MB-231 cells which were transfected with empty vector or PKCζ DN constructs(b) or incubated with DMSO, 500 nM wortmannin or 10 μM LY294002 (c). These cells were treated with 20 μM C 6 nanoliposome for 30 min before being injected into parallel plate chamber for cell detachment assays. The percentage of cells remaining attached to fibrinogen after each shear step was determined (expressed as % cells remaining bound). Results were expressed as mean ± SEM from three independent experiments.* p < 0.05 compared with NaL-C 6 +vector+scramble at each shear rate. vec, vector; scr, scrambled siRNA; siR, integrin α v β 3 siRNA. (b) Modulation of integrin α v β 3 affinity was required for NaL-C 6 -mediated and PKCζ-dependent cell adhesion weakening. The ability of MDA-MB-231 cells to bind to ligand-mimetic antibody Fab WOW-1 (10 ug/ml)in the presence or absence of 1 mM CaCl 2 or 250 μM MnCl 2 were analyzed by flow cytometry. The mean fluorescence intensity of staining was measured from three experiments. Results were expressed as mean ± SEM. ** p < 0.01 compared with normal; †† p < 0.01 compared with calcium. Untransfected or PKCζ DN-transfected MDA-MB-231 cells were incubated with 20 μM C 6 nanoliposome for 30 min before being subject to cell detachment assay in the presence or absence of 1 mM CaCl 2 or 250 μM MnCl 2 . * p < 0.05 compared with C 6 ; † p < 0.05 compared with NaL-C 6 +PKCζ DN.

Article Snippet: Mouse IgG anti-human α v β 3 (anti-CD51/61, clone 23C6), CD44H, VLA-4 and ICAM-1 (clone BBIG-I1) were purchased from R&D Systems (Minneapolis, MN).

Techniques: Shear, Adhesive, Transfection, Plasmid Preparation, Construct, Incubation, Injection, Flow Cytometry, Fluorescence, Staining