Journal: Cell Adhesion & Migration

Article Title: Recombinant disintegrin domain of human ADAM9 inhibits migration and invasion of DU145 prostate tumor cells

doi: 10.4161/19336918.2014.994917

Figure Lengend Snippet: rADAM9D promotes DU145 cell adhesion. Collagen type I (Col.I), collagen type IV (Col.IV), laminin, fibronectin (10 µg) (positive controls) or different quantities of ADAM9D (5, 10 and 50μg) were immobilized in the wells of a 96-well plate in adhesion buffer at 4°C. After blocking with 1% BSA (negative control), CMFDA-labeled DU145 cells (1 × 105 cells/well) were seeded in the wells. The plates were incubated at 37 °C for 30 min, washed, lysed and read for the release of fluorescence. BSA was used as negative control for cell adhesion. The results were obtained from 3 independent experiments in triplicate. The means that are significantly different from those of cells growing on collagen using ANOVA followed by post hoc Dunnett's test were shown by *(P ≤ 0.001). The results were normalized by the collagen type I values in each experiment. The error bars show the SE of three samples with less deviation from the mean.

Article Snippet: Cell line and culture DU145 human prostate tumor cell line was obtained from ATCC and maintained at 37°C in 5% CO 2 in RPMI culture medium (Cultilab) containing 10% FBS, penicillin (100 UI/ml), streptomycin (100 μg/ml) and L-glutamine (2mM).

Techniques: Blocking Assay, Negative Control, Labeling, Incubation, Fluorescence

Journal: Cell Adhesion & Migration

Article Title: Recombinant disintegrin domain of human ADAM9 inhibits migration and invasion of DU145 prostate tumor cells

doi: 10.4161/19336918.2014.994917

Figure Lengend Snippet: rADAM9D binds to DU145 through β1, α6, αvβ5 and αvβ3 integrins as demonstrated by an antibody competition assay (A) and by flow cytometry analysis (B). The integrin content of DU145 cell line was assessed by flow cytometry (C). (A) For antibody competition assay CMFDA-labeled cells were incubated with different anti-integrin antibodies (β1, α6, αvβ5, αvβ3, α2 and α4, at 10 µg/ml) and IgG control (10 µg/ml) before being plated on rADAM9D-coated (10 µg) wells. DU145 directly plated on rADAM9D or IgG-coated wells, without previous incubation with any antibody was used as positive control and BSA was used as negative control. (B) The integrin content of DU145 cells was determined by flow cytometry. Cells (1 × 105) were incubated for 40 min at 4°C with the specific antibodies cited earlier or control IgG. Cells were washed and incubated with secondary antibody labeled with FITC, at same conditions described before, washed and fixed with FACs buffer contaning 1% phormaldehyde overnight at 4°C. C. To verify the interaction with integrins, rADAM9D (1 µM) was previously incubated (30 min at room temperature) with DU145 cells, before the addition of antibodies. Cells were analyzed in FACSCanto. The results were obtained from 3 independent experiments in triplicate. The error bars show the SE of three samples with less deviation from the mean. The means that are significantly different from rADAM9D and IgG-coated wells using ANOVA followed of post hoc Dunnett's test were shown by *(P ≤ 0.001).

Article Snippet: Cell line and culture DU145 human prostate tumor cell line was obtained from ATCC and maintained at 37°C in 5% CO 2 in RPMI culture medium (Cultilab) containing 10% FBS, penicillin (100 UI/ml), streptomycin (100 μg/ml) and L-glutamine (2mM).

Techniques: Competitive Binding Assay, Flow Cytometry, Labeling, Incubation, Control, Positive Control, Negative Control

Journal: Cell Adhesion & Migration

Article Title: Recombinant disintegrin domain of human ADAM9 inhibits migration and invasion of DU145 prostate tumor cells

doi: 10.4161/19336918.2014.994917

Figure Lengend Snippet: rADAM9D inhibits the adhesion of DU145 to laminin but not to collagen type I. Ninety six-well plates were coated with laminin (A) or collagen type I (B) (10 µg/well) in adhesion buffer or 0.1% acetic acid, respectively, overnight at 4°C. After blocking with 1% BSA, CMFDA-labeled cells (1 × 105 cells/well) were incubated with different concentrations (100, 500, 1000nM) of rADAM9D and seeded in the wells. The plates were incubated at 37°C for additional 30 min. After washing, remaining cells were lysed, and the plate was read for the release of fluorescence. The results were obtained from 3 independent experiments and in triplicate. The results for rADAM9D were normalized by the collagen or laminin values in each experiment. The error bars show the SE of three samples with less deviation from the mean. The means for all rADAM9 concentrations were significantly different from the laminin using ANOVA followed by post hoc Dunnett's test: *(P ≤ 0.05), **(P ≤ 0.01) and ***(P ≤ 0.001).

Article Snippet: Cell line and culture DU145 human prostate tumor cell line was obtained from ATCC and maintained at 37°C in 5% CO 2 in RPMI culture medium (Cultilab) containing 10% FBS, penicillin (100 UI/ml), streptomycin (100 μg/ml) and L-glutamine (2mM).

Techniques: Blocking Assay, Labeling, Incubation, Fluorescence

Journal: Cell Adhesion & Migration

Article Title: Recombinant disintegrin domain of human ADAM9 inhibits migration and invasion of DU145 prostate tumor cells

doi: 10.4161/19336918.2014.994917

Figure Lengend Snippet: rADAM9D inhibits the invasion of DU145 cells through matrigel. DU145 cells (1.25 × 105 cells/ml) were seeded on the inserts (12 well-plate) of the invasion chamber in the presence or absence of rADAM9D (1 µM). Complete medium was used as a chemoattractant at the lower chamber. Plates were incubated for 22 h at 37°C and 5% CO2. Non-invading cells were removed with a cotton swab from the upper surface of the membrane and invading cells were fixed and 10 random fields from microscope slides were photographed and cells were counted using Image J software. Positive control (+ control) was made in the presence of chemoattractant (10% FBS) at the lower chamber and negative control (− control) was FBS free medium at the lower chamber. The results were obtained from 3 independent experiments in triplicate. The error bars show the SE of three samples with less deviation from the mean. The means that are significantly different from the positive control using ANOVA followed by post hoc Dunnett's test: *(P ≤ 0.001).

Article Snippet: Cell line and culture DU145 human prostate tumor cell line was obtained from ATCC and maintained at 37°C in 5% CO 2 in RPMI culture medium (Cultilab) containing 10% FBS, penicillin (100 UI/ml), streptomycin (100 μg/ml) and L-glutamine (2mM).

Techniques: Incubation, Membrane, Microscopy, Software, Positive Control, Control, Negative Control

Journal: Cell Adhesion & Migration

Article Title: Recombinant disintegrin domain of human ADAM9 inhibits migration and invasion of DU145 prostate tumor cells

doi: 10.4161/19336918.2014.994917

Figure Lengend Snippet: rADAM9D inhibits the migration of DU145 cells in a wound healing assay. (A) Cells (1 × 105 cells/ml) were plated in 24-wells plates and incubated properly until the culture reached 100% of confluence. Afterwards, a straight scratch was made with a pipette tip and cells were washed with culture medium to remove unbound cells and debris. Cells were incubated with ADAM9D (100, 500, 1000 and 2000 nM) for 24 h and 48 h. Only pictures from rADAMD9 500 and 2000 nM are represented. Central field was photographed at 0h, 24 h and 48 h (when cells close the scratch completely). (B) Closure area of migrating cells was measure using ImageJ software, and it was calculated the percentage of wound closure, comparing time zero and 24 hour. Results are expressed as percent of wound closure relative to control (untreated) cells. The results were obtained from 3 independent experiments in triplicate. The error bars show the SE of three samples with less deviation from the mean. The means that are significantly different from the control using ANOVA followed by post hoc Dunnett's test: *(P ≤ 0.05), **(P ≤ 0.01) and ***(P ≤ 0.001).

Article Snippet: Cell line and culture DU145 human prostate tumor cell line was obtained from ATCC and maintained at 37°C in 5% CO 2 in RPMI culture medium (Cultilab) containing 10% FBS, penicillin (100 UI/ml), streptomycin (100 μg/ml) and L-glutamine (2mM).

Techniques: Migration, Wound Healing Assay, Incubation, Transferring, Software, Control