metastatic human prostate cancer cell line pc3  (ATCC)

Journal: Journal of Extracellular Vesicles

Article Title: Tetraspanin CD82 interaction with cholesterol promotes extracellular vesicle–mediated release of ezrin to inhibit tumour cell movement

doi: 10.1080/20013078.2019.1692417

Figure Lengend Snippet: Effects of CD82 and its cholesterol binding on ERM proteins, the plasma membrane–actin cytoskeleton connectors. (a) Western blot analysis of total and phosphorylated ERM (Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558)) proteins in Du 145 transfectant cells. For cell lysis in suspension, the cells were detached and spun down, and cell pellets were lysed with RIPA buffer. For cell lysis in situ , the cells attached to culture dishes were directly lysed with RIPA buffer. (b) Total and phosphorylated ERM (Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558)) proteins in PC3, HT1080, and LnCap transfectants were examined with Western blot. The cells were lysed directly in culture dishes. (c) Regulation of Ezrin distribution by CD82 or its cholesterol binding. Cells cultured on glass coverslips for 4–5 days were fixed, permeabilised and incubated with Ezrin Ab and the secondary Ab , phalloidin, and DAPI, and examined with confocal microscopy. Scale bar: 10 µm. (d) Releases of ERM proteins via EVs. Exosomes isolated from culture supernatants of the cells were lysed with RIPA buffer, equal amounts of EV proteins (5 μg/lane) were loaded to SDS–PAGE, and ERM and CD9 proteins were examined in Western blot. (e)–(f) The analyses were performed as described in (c). (g) Du145-CD82 WT transfectant cells were cultured on glass coverslips in the serum-free DMEM containing either DMSO (0.1% v/v), TAK-475 (20 µM) or cholesterol (15 µg/ml)/BSA(2%) for 48 h, fixed, stained for Ezrin and F-actin, and photographed with confocal microscopy. Scale bar: 10 µm. Extracellular Ezrin proteins were quantified with ImageJ and presented as relative fluorescence intensity (mean ± SEM, n = 3 individual experiments). *: p < 0.05, **: p < 0.01 and ***: p < 0.001. (h) Cells were treated with DMSO (0.1% v/v) or TAK-475 (20 µM) in serum-free DMEM for 48 h, detached with 2 mM EDTA/PBS and lysed with RIPA buffer. Cell lysates were examined for Ezrin and actin in Western blot.

Article Snippet: Du145 and PC3 human metastatic prostate cancer cell lines, LnCap human prostate cancer cell line and HT1080 human fibroblastoma cell line were obtained from ATCC (VA) and cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Corning) supplemented with 10% foetal bovine serum (FBS) (Biowest, TX), penicillin and streptomycin.

Techniques: Binding Assay, Clinical Proteomics, Membrane, Western Blot, Transfection, Lysis, Suspension, In Situ, Cell Culture, Incubation, Confocal Microscopy, Isolation, SDS Page, Staining, Fluorescence

Journal: bioRxiv

Article Title: miR-26a regulates extracellular vesicle secretion from prostate cancer cells via targeting SHC4, PFDN4 and CHORDC1

doi: 10.1101/646380

Figure Lengend Snippet: A. Venn diagram of predicted miR-26a targets (TargetScan) and transcripts that were experimentally repressed >2-fold by miR-26a overexpression in prostate cancer cells (PC3M or PC3) relative to control conditions. B. Schematic of the high-throughput compatible EV biogenesis assay to choose EV biogenesis-regulating genes. C. Venn diagram showing genes that suppress EV secretion evaluated by ExoScreen. The genes whose relative EV secretion/cell viability was lower than that of miR-26a plus 0.3 were selected in each assay. The secretion of EV was evaluated by ExoScreen, and the cell viability was measured by the MTS assay. D. The effect of siRNAs against candidate genes on EV secretion in PC3M cells. The EV secretion per cell was evaluated by the signal intensity of ExoScreen per cell. The values are depicted as the fold change relative to the negative control siRNA (control). The values are the mean±SE (n=3). *, p<0.05; **, p<0.01; and n.s., not significant. E. The effect of siRNAs against candidate genes on EV secretion per PC3M cell. The particle number of EVs was measured using a nanoparticle tracking system. The values are the mean±SE (n=3). *, p<0.05; and n.s., not significant. F. The effect of SHC4, PFDN4 and CHORDC1 siRNA on the mRNA expression level of each gene. β-actin was used as an internal control. Error bars represent the s.e. deduced by Student’s t-test (*P<0.05, **<0.01). n.s., no significant difference. The data are representative of at least three independent experiments. The values are the mean±SE (n=3). **, p<0.01.

Article Snippet: The human prostate cancer epithelial metastatic cell line PC3 (ATCC CRL-1435) was purchased from ATCC.

Techniques: Over Expression, Control, High Throughput Screening Assay, MTS Assay, Negative Control, Expressing

Journal: Molecular cancer research : MCR

Article Title: Agonist-induced CXCR4 and CB2 Heterodimerization Inhibits Gα13/RhoA-mediated Migration

doi: 10.1158/1541-7786.MCR-16-0481

Figure Lengend Snippet: (A) Serum-starved PC3 cells were pre-treated for 1 hour with Y-27632 or AMD3100 followed by treatment with CXCL12 for 1 minute. Alternatively, PC3 cells were treated for 1 minute with CXCL12 or AM1241 alone, or CXCL12/AM1241 simultaneously. Lysates were incubated with Rhotekin-RBD beads prior to harvesting for immunoblotting for RhoA-GTP protein expression; total RhoA represents lysate prior to pull-down with Rhotekin-RBD beads. (B) PC3 cells, (C) MDA-MB-231 cells and (D) HEK 293T cells were treated as described in Methods, then lysates were incubated with anti-Gα13 antibody overnight at 4°C followed by standard immunoprecipitation techniques. Isolated protein lysates was immunoblotted for RhoA protein expression; input represents lysate prior to immunoprecipitation. (E) PC3 were transfected with 60μM human siRNA targeting CXCR4 or non-specific control siRNA for 12 hours, followed by recovery in 10%FBS/RPMI for 24 hours. Cells were then harvested for Gα13/RhoA immunoprecipitation or western blotting. Total ERK1/2 and AKT were used as: (i) loading input controls to demonstrate that siRNA was specific; and (ii) to demonstrate that equal amounts of protein were used for assay. (F) Serum-starved PC3 cells were treated as described above, and whole protein lysates were immunoblotted for PRG protein expression; α-tubulin was used as a loading control. Each experiment was performed at least twice. *p<0.05 and **p<0.001

Article Snippet: Cell lines and reagents Human metastatic prostate cancer cell line (PC3), human metastatic breast cancer cell line (MDA-MB-231) and human embryonic kidney cells (HEK 293T) were purchased from American Type Culture Collection (ATCC).

Techniques: Incubation, Western Blot, Expressing, Immunoprecipitation, Isolation, Transfection, Control

Journal: Molecular cancer research : MCR

Article Title: Agonist-induced CXCR4 and CB2 Heterodimerization Inhibits Gα13/RhoA-mediated Migration

doi: 10.1158/1541-7786.MCR-16-0481

Figure Lengend Snippet: Serum-starved PC3 cells were pre-treated for 1 hour with Y-27632 or AMD3100 prior to treatment with CXCL12 for 90 minutes. Subsequently, cells were treated for 90 minutes with CXCL12 or AM1241 alone, or CXCL12/AM1241 simultaneously. Samples were fixed and stained with FITC-phalloidin to visualize F-actin; nuclei were counter-stained with DAPI. Arrows highlight areas of lamellae formation and membrane ruffling.

Article Snippet: Cell lines and reagents Human metastatic prostate cancer cell line (PC3), human metastatic breast cancer cell line (MDA-MB-231) and human embryonic kidney cells (HEK 293T) were purchased from American Type Culture Collection (ATCC).

Techniques: Staining, Membrane

Journal: Molecular cancer research : MCR

Article Title: Agonist-induced CXCR4 and CB2 Heterodimerization Inhibits Gα13/RhoA-mediated Migration

doi: 10.1158/1541-7786.MCR-16-0481

Figure Lengend Snippet: (A) Serum-starved HEK 293T cells were pre-treated for 1 hour with Y-27632 or AMD3100 prior to treatment with CXCL12 for 90 minutes. Subsequently, cells were treated for 90 minutes with CXCL12 or AM1241 alone, or CXCL12/AM1241 simultaneously. Samples were fixed and stained with FITC-phalloidin to visualize F-actin; nuclei were counter-stained with DAPI. Arrows highlight areas of lamellae formation and membrane ruffling. (B–D) Serum-starved PC3 cells were pre-treated with Y-27632 or AMD3100 followed by treatment with CXCL12, AM1241 or agonists simultaneously for 90 minutes. Whole protein lysates were analyzed for (B) F-actin, (C) pMLC or (D) LKB1. Each experiment was performed at least twice.

Article Snippet: Cell lines and reagents Human metastatic prostate cancer cell line (PC3), human metastatic breast cancer cell line (MDA-MB-231) and human embryonic kidney cells (HEK 293T) were purchased from American Type Culture Collection (ATCC).

Techniques: Staining, Membrane

Journal: Molecular cancer research : MCR

Article Title: Agonist-induced CXCR4 and CB2 Heterodimerization Inhibits Gα13/RhoA-mediated Migration

doi: 10.1158/1541-7786.MCR-16-0481

Figure Lengend Snippet: (A–B) Serum-starved PC3 cells were pre-treated for 1 hour with Y-27632 or AMD3100 followed by treatment with CXCL12 and/or AM1241 for up to 6 hours as described in Methods. ImageJ was used to measure the width of each wound at 0 hour and 6 hour. Experiments were performed thrice. *p<0.05 and **p<0.001. (C–D) 5x104 cells each were seeded into the upper transwell chamber and allowed to migrate towards combinations of agonists and chemical inhibitors in the lower chamber for 6 hours at 37ºC, 5% CO2. Five fields of each transwell insert were randomly selected and counted for migrated cells at 10X magnification using a Zeiss Axiovert 200M light microscope, and results were analyzed via GraphPad Prism. Experiments were repeated thrice, and data represents the average of three independent experiments. (D) A graphical representation of total migrated cells. Data represents the average of 3 independent experiments; *p<0.05 and **p<0.001. (E) HUVECs were seeded in the upper chamber of transwell inserts allowing for the formation of a confluent monolayer. Fluorescently-labeled, serum-starved PC3 cells were added on top of HUVECs in the upper chamber, then combinations of agonists and chemical inhibitors were added to the lower (bottom) chambers followed by a 24 hour incubation. Fluorescent invasive cells were quantified at OD 480 nm/520 nm. Experiments were performed thrice *p<0.05 and **p<0.001.

Article Snippet: Cell lines and reagents Human metastatic prostate cancer cell line (PC3), human metastatic breast cancer cell line (MDA-MB-231) and human embryonic kidney cells (HEK 293T) were purchased from American Type Culture Collection (ATCC).

Techniques: Light Microscopy, Labeling, Incubation

Journal: Molecular cancer research : MCR

Article Title: Agonist-induced CXCR4 and CB2 Heterodimerization Inhibits Gα13/RhoA-mediated Migration

doi: 10.1158/1541-7786.MCR-16-0481

Figure Lengend Snippet: (A) Serum-starved PC3 cells were pre-treated for 1 hour with Y-27632 or AMD3100 followed by treatment with CXCL12 or AM1241 alone, or CXCL12/AM1241 simultaneously for 6 hours. Cell lysates were harvested for integrin α5 and integrin β3 protein expression; α-tubulin was used as a loading control. Experiment was performed at least twice. (B–D) Cells were serum-starved and pre-treated Y-27632 and AMD3100 for 1 hour prior to the addition of CXCL12 or AM1241 alone, or CXCL12/AM1241 simultaneously on matrix-coated plates for 2 hours, stained with Cell Stain Solution (Cell Bio Labs, Inc.), and then analyzed at OD 560nm. Each assay was performed at least thrice. *p<0.05 and **p<0.001.

Article Snippet: Cell lines and reagents Human metastatic prostate cancer cell line (PC3), human metastatic breast cancer cell line (MDA-MB-231) and human embryonic kidney cells (HEK 293T) were purchased from American Type Culture Collection (ATCC).

Techniques: Expressing, Control, Staining