human prostate tumor derived cell line du145  (ATCC)

Journal: ACS Omega

Article Title: Novel C-2 Aromatic Heterocycle-Substituted Triterpenoids Inhibit Hedgehog Signaling in GLI1 Overexpression Cancer Cells

doi: 10.1021/acsomega.4c11479

Figure Lengend Snippet: Effect of compounds 11a and 11b on the protein expression of the Gli1 transcription factor and its transcriptional targets in A549 (A) and DU145 (B) cells following 24 h of incubation. An antibody against GAPDH was used as an internal loading control.

Article Snippet: The human prostate tumor-derived cell line DU145 was obtained from ATCC (American Type Culture Collection) and cultured in RPMI 1640 (Roswell Park Memorial Institute) medium supplemented with 10% (v/v) fetal bovine serum (FBS).

Techniques: Expressing, Incubation, Control

Journal: Pharmaceutics

Article Title: The Antitumoral/Antimetastatic Action of the Flavonoid Brachydin A in Metastatic Prostate Tumor Spheroids In Vitro Is Mediated by (Parthanatos) PARP-Related Cell Death

doi: 10.3390/pharmaceutics14050963

Figure Lengend Snippet: ( A , B ) Morphology and integrity of DU145 spheroids after treatment with Brachydin A (BrA) for 0 (Day 4), 72 (Day 7), 120 (Day 9), and 168 h (Day 11). All images were obtained using the Axio Cam MRc capture system coupled to Axio LabA1 microscope, using the 10× ( A ) and 40× ( C ) objectives. ( C ) Volume (area/µm 3 ) of DU145 spheroids after 0 (Day 4), 72 (Day 7), 120 (Day 9), and 168 h (Day 11) of exposure with BrA and respective controls. ( D ) Survival fraction of DU145 cells disaggregated from spheroids treated for 72 h with BrA and their respective controls. All data points from volume/morphology represent the mean of six ( n = 6) spheroids/replicates analyzed in three biological experiments ( n = 3). * Values statistically different from the NC group at the point (date) (* p < 0.05; ** p < 0.01; *** p < 0.001; ANOVA followed by Dunnett’s post-test). NC: negative control (RPMI 1640); SC: solvent control (1% DMSO); DTX: Docetaxel (50 μM; positive control). Scale: 200 μm.

Article Snippet: The prostate tumor DU145 cell line, isolated from a metastatic brain site, was purchased from ATCC ® (American Type Culture Collection—Cat.

Techniques: Microscopy, Negative Control, Solvent, Control, Positive Control

Journal: Pharmaceutics

Article Title: The Antitumoral/Antimetastatic Action of the Flavonoid Brachydin A in Metastatic Prostate Tumor Spheroids In Vitro Is Mediated by (Parthanatos) PARP-Related Cell Death

doi: 10.3390/pharmaceutics14050963

Figure Lengend Snippet: ( A , B ) Photomicrographs from DU145 spheroids in ECM gel obtained after 0, 24, and 48 h of exposure with Brachydin A (BrA) and respective controls. All images were acquired using the Axio Cam MRc capture system coupled to Axio LabA1 microscope, using the 10× objective, and analyzed using the AxioVision 3.1 software. Scale: 200 μm. ( C , D ) Cell migration area (µm 3 ) from DU-145 spheroids after 0, 24, and 48 h exposures with BrA and their respective controls. All points/bars represent the mean ± standard deviation of the covered area from six ( n = 6) spheroids/replicates in three biological experiments ( n = 3). * Values statistically different from the NC group at the time-point (date) (** p < 0.01; *** p < 0.001; ANOVA followed by Dunnett’s post-test). NC: negative control (RPMI 1640); SC: solvent control (1% DMSO); DTX: Docetaxel (50 μM; positive control).

Article Snippet: The prostate tumor DU145 cell line, isolated from a metastatic brain site, was purchased from ATCC ® (American Type Culture Collection—Cat.

Techniques: Microscopy, Software, Migration, Standard Deviation, Negative Control, Solvent, Control, Positive Control

Journal: Pharmaceutics

Article Title: The Antitumoral/Antimetastatic Action of the Flavonoid Brachydin A in Metastatic Prostate Tumor Spheroids In Vitro Is Mediated by (Parthanatos) PARP-Related Cell Death

doi: 10.3390/pharmaceutics14050963

Figure Lengend Snippet: ( A , B ) Photomicrographs of DU145 spheroids in bovine skin gelatin after 0 and 72 h of exposure with Brachydin A (BrA) and respective controls. The images were acquired using the Axio Cam MRc image capture system coupled to an Axio LabA1 microscope, using the 10× objective, and analyzed using the AxioVision 3.1 software. Scale: 200 μm. ( C ) Percentage (%) of cell invasion (invadopodia) formed by DU145 spheroids after 24, 48, and 72 h of exposure with BrA and respective controls. The invasion area (µm 3 ) into the ECM (gelatin) was converted to % considering the area increase at 0 h. All data points represent the mean ± standard deviation of four ( n = 4) spheroids/replicates in three biological experiments ( n = 3). * Values statistically different from the NC group at that point (date) (* p < 0.05; ** p < 0.01; *** p < 0.001; ANOVA followed by Dunnett’s post-test). NC: negative control (RPMI 1640); SC: solvent control (1% DMSO); DTX: Docetaxel (50 μM; positive control). Scale: 200 μm.

Article Snippet: The prostate tumor DU145 cell line, isolated from a metastatic brain site, was purchased from ATCC ® (American Type Culture Collection—Cat.

Techniques: Microscopy, Software, Standard Deviation, Negative Control, Solvent, Control, Positive Control

Journal: Pharmaceutics

Article Title: The Antitumoral/Antimetastatic Action of the Flavonoid Brachydin A in Metastatic Prostate Tumor Spheroids In Vitro Is Mediated by (Parthanatos) PARP-Related Cell Death

doi: 10.3390/pharmaceutics14050963

Figure Lengend Snippet: Flow cytometric analysis of DU145 spheroids treated with Brachydin A (BrA) for 72 h and respective controls and stained with Annexin V-FITC/propidium iodide (PI). ( A ) Percentage (%) of viable, early apoptotic, late apoptotic, and necrotic cells in DU145 spheroids after 72 h exposure with Brachydin A (BrA). The bars represent the mean ± standard deviation of eight ( n = 8) spheroids/replicates disaggregated in three biological experiments ( n = 3). *, # Values statistically different from the NC group ( p < 0.05; two-way ANOVA followed by Dunnett’s post-test). ( B ) Representative dot-plots of DU145 spheroids treated for 72 h with BrA. Lower left quadrant (Q3): negative cells for both Annexin V-FITC and PI; lower right quadrant (Q4): cells labeled with Annexin V (early apoptotic cells); upper left quadrant (Q1): cells labeled only with PI (necrotic cells); right upper quadrant (Q2): cells labeled with Annexin V and PI (late apoptotic cells). The bars represent the mean ± standard deviation of experiments with six ( n = 6) spheroids/replicates and three biological experiments ( n = 3). Values statistically different from the NC group for * early apoptosis; # late apoptosis and % necrosis ( p < 0.05; ANOVA followed by Dunnett’s post-test). NC: negative control (RPMI 1640); SC: solvent control (1% DMSO); DTX: Docetaxel (50 μM; positive control).

Article Snippet: The prostate tumor DU145 cell line, isolated from a metastatic brain site, was purchased from ATCC ® (American Type Culture Collection—Cat.

Techniques: Staining, Standard Deviation, Labeling, Negative Control, Solvent, Control, Positive Control

Journal: Pharmaceutics

Article Title: The Antitumoral/Antimetastatic Action of the Flavonoid Brachydin A in Metastatic Prostate Tumor Spheroids In Vitro Is Mediated by (Parthanatos) PARP-Related Cell Death

doi: 10.3390/pharmaceutics14050963

Figure Lengend Snippet: ( A , C , E , G ) Representative images of the effects of Brachydin A (BrA) and controls on mitochondrial ROS (MitoSOX Red), mitochondrial membrane potential (MitoStatus), apoptosis (Caspase 3/7), and necrosis (PI) in DU145 spheroids, respectively. The photomicrographs were acquired using the In-Cell Analyzer 2200 platform, using a 4× objective, in DAPI (Hoechst 33342), Cy5 (MitoStatus), FITC (CellEvent Caspase 3/7), and Cy3 (PI) channels. Scale: 200 µm. ( B , D , F , H ) Fluorescence intensity for MitoSOX Red (TexasRed), MitoStatus Red (Cy5), Caspase 3/7 (FITC), and PI (Cy3) in DU145 spheroids after treatment with BrA and respective controls. Data were normalized to the mean fluorescence intensity of the NC group at each time/point. The bars represent the mean ± standard deviation of four ( n = 4) spheroids/replicates from three biological experiments ( n = 3). * Values statistically different from the NC group (* p < 0.05; ** p < 0.01; *** p < 0.001; ANOVA followed by Dunnett’s post-test). NC: negative control (RPMI 1640); SC: solvent control (1% DMSO); DTX: Docetaxel (50 µM; positive control).

Article Snippet: The prostate tumor DU145 cell line, isolated from a metastatic brain site, was purchased from ATCC ® (American Type Culture Collection—Cat.

Techniques: Membrane, Fluorescence, Standard Deviation, Negative Control, Solvent, Control, Positive Control

Journal: Pharmaceutics

Article Title: The Antitumoral/Antimetastatic Action of the Flavonoid Brachydin A in Metastatic Prostate Tumor Spheroids In Vitro Is Mediated by (Parthanatos) PARP-Related Cell Death

doi: 10.3390/pharmaceutics14050963

Figure Lengend Snippet: Western blotting analysis of protein expression and densitometric quantifications in DU145 spheroids treated with BrA for 24 h ( A , B ) and 72 h ( C , D ). Protein expression was normalized with α-Tubulin (24 h) and β-Actin (72 h) from the SC group. All bars are presented as mean ± standard deviation of experiments performed with ninety-six ( n = 96) spheroids/replicates and two biological experiments ( n = 2). * Statistically different values from the SC group ( p < 0.05; ANOVA followed by Dunnett’s post-test). SC: solvent control (1% DMSO).

Article Snippet: The prostate tumor DU145 cell line, isolated from a metastatic brain site, was purchased from ATCC ® (American Type Culture Collection—Cat.

Techniques: Western Blot, Expressing, Standard Deviation, Solvent, Control

Journal: Pharmaceutics

Article Title: The Antitumoral/Antimetastatic Action of the Flavonoid Brachydin A in Metastatic Prostate Tumor Spheroids In Vitro Is Mediated by (Parthanatos) PARP-Related Cell Death

doi: 10.3390/pharmaceutics14050963

Figure Lengend Snippet: A proposed model of Brachydin A (BrA) effects in mPCa (DU145) tumor spheroids. BrA initially causes mitochondrial dysfunction and DNA fragmentation by PARP overactivation. Subsequently, cleaved-PARP promotes apoptosis/necrosis mixed-effects, DNA damage, and inflammatory response, suggesting that parthanatos cell death mediates the antiproliferative, cytotoxic, and antimetastatic properties observed in DU145 spheroids.

Article Snippet: The prostate tumor DU145 cell line, isolated from a metastatic brain site, was purchased from ATCC ® (American Type Culture Collection—Cat.

Techniques:

Journal: Cancers

Article Title: Insulin-Like Growth Factor-1 Influences Prostate Cancer Cell Growth and Invasion through an Integrin α3, α5, αV, and β1 Dependent Mechanism

doi: 10.3390/cancers14020363

Figure Lengend Snippet: IGF-1R (IGF1R) and pIGF-1R (pIGF1R) expression levels in DU145, PC3, LNCaP and VCaP cells. Light gray: isotype control; dark gray: specific fluorescence. Flow cytometry curves are representative for one of three experiments.

Article Snippet: The human prostate tumor cell lines DU145, PC3, and LNCaP were obtained from DSMZ (Braunschweig, Germany).

Techniques: Expressing, Control, Fluorescence, Flow Cytometry

Journal: Cancers

Article Title: Insulin-Like Growth Factor-1 Influences Prostate Cancer Cell Growth and Invasion through an Integrin α3, α5, αV, and β1 Dependent Mechanism

doi: 10.3390/cancers14020363

Figure Lengend Snippet: DU145, PC3, LNCaP, and VCaP cell growth in response to IGF-1. DU145 and PC3 cells were grown in FBS free medium (0%), in medium containing 2% FBS (2%) or 10% FBS (10%). LNCaP and VCaP were cultivated in the presence of 10% FBS. Untreated cells served as controls. Cell number was evaluated after 24, 48, and 72 h by the MTT assay. Error bars indicate standard deviation. Experiments were repeated five times. One representative experiment is shown. * indicates p < 0.05, # indicates p < 0.01.

Article Snippet: The human prostate tumor cell lines DU145, PC3, and LNCaP were obtained from DSMZ (Braunschweig, Germany).

Techniques: MTT Assay, Standard Deviation

Journal: Cancers

Article Title: Insulin-Like Growth Factor-1 Influences Prostate Cancer Cell Growth and Invasion through an Integrin α3, α5, αV, and β1 Dependent Mechanism

doi: 10.3390/cancers14020363

Figure Lengend Snippet: ( A ) Adhesion of DU145 and PC3 cells to immobilized collagen or fibronectin after stimulation with IGF-1 for 4 or 24 h. Mean number of adherent tumor cells from five fields. * indicates significant up-regulation to untreated control, # indicates significant down-regulation to untreated control ( n = 4). ( B ) Chemotactic movement of DU145 and PC3 cells after stimulation with IGF-1 for 4 or 24 h. Controls remained untreated. Mean number of tumor cells crawling beneath the filter membrane. * indicates significant up-regulation to untreated control, # indicates significant down-regulation to untreated control ( n = 3). All values are related to untreated controls set to 100%.

Article Snippet: The human prostate tumor cell lines DU145, PC3, and LNCaP were obtained from DSMZ (Braunschweig, Germany).

Techniques: Control, Membrane

Journal: Cancers

Article Title: Insulin-Like Growth Factor-1 Influences Prostate Cancer Cell Growth and Invasion through an Integrin α3, α5, αV, and β1 Dependent Mechanism

doi: 10.3390/cancers14020363

Figure Lengend Snippet: Motile crawling of DU145 and PC3 cells treated with IGF-1 (versus controls). The upper left side shows photomicrographs of the DU145 scratch assay taken after 0 (start), 8, 10, and 12 h (related to 4 h IGF-1 pre-stimulation). The upper right side shows the results from the quantitative calculation expressed as relative wound density (4 and 24 h pre-stimulation). DU145 were grown in 2% FBS. Lower panels: Relative wound density of DU145 cells following 4 or 24 h IGF-1 pre-stimulation and cultivation in 10% FBS, and relative wound density of PC3 cells stimulated with IGF-1 for 24 h and cultured in 2 or 10% FBS. Mean values of three experiments are shown. * indicates significant up-regulation compared to untreated controls ( n = 3).

Article Snippet: The human prostate tumor cell lines DU145, PC3, and LNCaP were obtained from DSMZ (Braunschweig, Germany).

Techniques: Wound Healing Assay, Cell Culture

Journal: Cancers

Article Title: Insulin-Like Growth Factor-1 Influences Prostate Cancer Cell Growth and Invasion through an Integrin α3, α5, αV, and β1 Dependent Mechanism

doi: 10.3390/cancers14020363

Figure Lengend Snippet: Surface expression of integrin α and β subtypes on DU145, PC3, LNCaP, and VCaP cells. Counts indicate cell number; fluorescence is expressed by mean fluorescence units (MFU). One representative of three separate experiments is shown. Solid line = specific fluorescence, dotted line = isotype control.

Article Snippet: The human prostate tumor cell lines DU145, PC3, and LNCaP were obtained from DSMZ (Braunschweig, Germany).

Techniques: Expressing, Fluorescence, Control

Journal: Cancers

Article Title: Insulin-Like Growth Factor-1 Influences Prostate Cancer Cell Growth and Invasion through an Integrin α3, α5, αV, and β1 Dependent Mechanism

doi: 10.3390/cancers14020363

Figure Lengend Snippet: IGF-1 stimulated surface expression of the integrins α3, α5, αV, and β1 on DU145 and PC3 cells, evaluated after 2, 4 and 24 h. All values are related to untreated controls. MFU: Mean fluorescence units. * indicates significant difference to controls ( n = 3).

Article Snippet: The human prostate tumor cell lines DU145, PC3, and LNCaP were obtained from DSMZ (Braunschweig, Germany).

Techniques: Expressing, Fluorescence

Journal: Cancers

Article Title: Insulin-Like Growth Factor-1 Influences Prostate Cancer Cell Growth and Invasion through an Integrin α3, α5, αV, and β1 Dependent Mechanism

doi: 10.3390/cancers14020363

Figure Lengend Snippet: Left side: Protein profile of integrins α3, α5, αV, and β1. DU145 cells were either stimulated with IGF-1 for 24 h or exposed to culture medium without IGF-1 (Ctrl). One representative of three separate experiments is shown. Each protein analysis was accompanied by a β-actin loading control. One representative internal control is shown. Right side: Pixel density analysis of the protein expression level of DU145 cells stimulated with IGF. The ratio of protein intensity/β-actin intensity is expressed as percentage of controls, indicated by line at 100%. * indicates significant difference to controls.

Article Snippet: The human prostate tumor cell lines DU145, PC3, and LNCaP were obtained from DSMZ (Braunschweig, Germany).

Techniques: Control, Expressing

Journal: Cancers

Article Title: Insulin-Like Growth Factor-1 Influences Prostate Cancer Cell Growth and Invasion through an Integrin α3, α5, αV, and β1 Dependent Mechanism

doi: 10.3390/cancers14020363

Figure Lengend Snippet: Left: Protein profile of cell signaling proteins. DU145 cells were either stimulated with IGF for 24 h or received culture medium without IGF (Ctrl). One representative of three separate experiments is shown. Each protein analysis was accompanied by a β-actin loading control. One representative internal control is shown. Right: Pixel density analysis of the protein expression level of DU145 cells stimulated with IGF. The ratio of protein intensity/β-actin intensity is expressed as percentage of controls set to 100%. * indicates significant difference to controls.

Article Snippet: The human prostate tumor cell lines DU145, PC3, and LNCaP were obtained from DSMZ (Braunschweig, Germany).

Techniques: Control, Expressing

Journal: Cancers

Article Title: Insulin-Like Growth Factor-1 Influences Prostate Cancer Cell Growth and Invasion through an Integrin α3, α5, αV, and β1 Dependent Mechanism

doi: 10.3390/cancers14020363

Figure Lengend Snippet: Left: protein profile of integrins α3, α5, αV, and β1 in PC3 cells. Right: protein profile of cell signaling proteins in PC3 cells. DU145 or PC3 cells were either stimulated with IGF for 24 h or received culture medium without IGF (Ctrl). One representative of three separate experiments is shown. Each protein analysis was accompanied by a β-actin loading control. One representative internal control is shown.

Article Snippet: The human prostate tumor cell lines DU145, PC3, and LNCaP were obtained from DSMZ (Braunschweig, Germany).

Techniques: Control

Journal: Cancers

Article Title: Insulin-Like Growth Factor-1 Influences Prostate Cancer Cell Growth and Invasion through an Integrin α3, α5, αV, and β1 Dependent Mechanism

doi: 10.3390/cancers14020363

Figure Lengend Snippet: Upper panels: cell growth dynamics of DU145, PC3, LNCaP, and VCaP cells after blockade through monoclonal antibodies against α3, α5, αV, or β1. Controls remained untreated. * indicates significant difference to untreated controls ( n = 4). Lower panels: chemotactic movement of DU145 and PC3 cells after blockade through monoclonal antibodies (specific blockade against α3, α5, αV, or β1). Controls remained untreated. All values are related to the untreated controls set to 100%. * indicates significant difference ( n = 3).

Article Snippet: The human prostate tumor cell lines DU145, PC3, and LNCaP were obtained from DSMZ (Braunschweig, Germany).

Techniques: Bioprocessing

Journal: Cancers

Article Title: Insulin-Like Growth Factor-1 Influences Prostate Cancer Cell Growth and Invasion through an Integrin α3, α5, αV, and β1 Dependent Mechanism

doi: 10.3390/cancers14020363

Figure Lengend Snippet: Adhesion modulation of DU145 and PC3 through monoclonal antibodies against α3, α5, αV, or β1 (left: adhesion to collagen; right: adhesion to fibronectin). Controls remained untreated. * indicates significant difference to untreated controls ( n = 3).

Article Snippet: The human prostate tumor cell lines DU145, PC3, and LNCaP were obtained from DSMZ (Braunschweig, Germany).

Techniques: Bioprocessing

Journal: Cancers

Article Title: Insulin-Like Growth Factor-1 Influences Prostate Cancer Cell Growth and Invasion through an Integrin α3, α5, αV, and β1 Dependent Mechanism

doi: 10.3390/cancers14020363

Figure Lengend Snippet: ( A ) Protein expression level following siRNA transfection (untreated control versus scrambled siRNA versus specific siRNA). Each protein analysis was accompanied by a β-actin loading control. One representative internal control is shown. ( B ) Cell growth of DU145 treated with an integrin α3, α5, αV, or FAK specific siRNA, evaluated by the MTT-assay. One representative of three separate experiments is shown. ( C ) Chemotactic movement of DU145 cells after knocking down integrin α3, α5, or αV. All values are related to the scrambled controls set to 100%. * indicates significant difference to controls ( n = 3).

Article Snippet: The human prostate tumor cell lines DU145, PC3, and LNCaP were obtained from DSMZ (Braunschweig, Germany).

Techniques: Expressing, Transfection, Control, MTT Assay

Journal: Cancers

Article Title: Insulin-Like Growth Factor-1 Influences Prostate Cancer Cell Growth and Invasion through an Integrin α3, α5, αV, and β1 Dependent Mechanism

doi: 10.3390/cancers14020363

Figure Lengend Snippet: ( A ) DU145 cell number in response to integrin blockade. Tumor cells were grown in 2 or 10% FBS and in the presence or without IGF-1. Untreated cells served as controls. Cell number was evaluated after 24, 48, and 72 h by the MTT assay. ( B ) Chemotactic movement of DU145 cells after blockade through monoclonal antibodies (specific blockade against α3, αV, or β1) with or without IGF-1 activation. ( C ) Adhesion modulation of DU145 through integrin β1 blockade in the presence of 2 versus 10% FBS. ( B , C ) are related to the non-blocked controls set to 100%. Error bars indicate standard deviation. Experiments were repeated three times. One representative experiment is shown. * indicates p < 0.05.

Article Snippet: The human prostate tumor cell lines DU145, PC3, and LNCaP were obtained from DSMZ (Braunschweig, Germany).

Techniques: MTT Assay, Bioprocessing, Activation Assay, Standard Deviation

Journal: Cancers

Article Title: Insulin-Like Growth Factor-1 Influences Prostate Cancer Cell Growth and Invasion through an Integrin α3, α5, αV, and β1 Dependent Mechanism

doi: 10.3390/cancers14020363

Figure Lengend Snippet: ( Left ) Integrin subtype and FAK expression level in DU145 cells following treatment with Akt specific siRNA (versus untreated controls or scrambled siRNA). ( Right ) mTOR signaling in DU145 cells following defactinib treatment for 24 and 72 h. Controls remained untreated. One representative of three separate experiments is shown. Each protein analysis was accompanied by a β-actin loading control. One representative internal control is shown.

Article Snippet: The human prostate tumor cell lines DU145, PC3, and LNCaP were obtained from DSMZ (Braunschweig, Germany).

Techniques: Expressing, Control

Journal: Nanotheranostics

Article Title: Perfluorocarbon nanodroplets can reoxygenate hypoxic tumors in vivo without carbogen breathing

doi: 10.7150/ntno.29908

Figure Lengend Snippet: Biodistribution of DiI-labeled PFOB nanodroplets in NRG mice bearing DU145 tumor xenografts: (A) Representative ex vivo fluorescence images of organs and tumor tissues acquired at 3h, 6h, and 24h post-injection of PFOB nanodroplets.) (B) The quantitative fluorescence signal of DiI acquired from organs and tumors tissues acquired at 3h, 6h, and 24h post-injection (n≥3, p-value <0.005 from Tukey test was shown as ***).

Article Snippet: An androgen-independent human prostate tumor cell line (DU145) was obtained from ATCC (Manassas, VA, USA).

Techniques: Labeling, Ex Vivo, Fluorescence, Injection

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: 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: 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: 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: 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: 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

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