Uthor Manuscript Author Manuscript Author Manuscript Author ManuscriptMATERIALS AND METHODSCell lines and cell culture Non-malignant epithelial prostate cell lines (RWPE-1 and PWR-1E) and prostate carcinoma cell lines (LNCaP, Du145, PC3) had been obtained from the American Type Culture Collection (ATCC) and cultured beneath recommended situations as described previously (28). RWPE-1 and PWR-1E cells have been cultured in keratinocyte growth medium supplemented with five ng/mL human recombinant epidermal growth issue, 0.05 mg/mL bovine pituitary extract (Invitrogen). LNCaP, Du145, PC3 were maintained in RPMI 1640 media supplemented with 10 fetal bovine serum (FBS) (Atlanta Epoxide Hydrolase Inhibitor Storage & Stability biologicals) and 1 penicillin/streptomycin. Cell lines had been maintained in an incubator with a humidified atmosphere of 95 air and five CO2 at 37 . Cell lines had been authenticated by DNA short-tandem repeat analysis by ATCC. The experiments with cell lines had been performed inside 6 months of their procurement/ resuscitation. miRNA transfections Cells had been plated in growth medium with no antibiotics 24hrs ahead of transfection. Transient transfection of miRNA precursor/anti-miR miRNA inhibitor (Ambion) was carried out applying Lipofectamine 2000 (Invitrogen) in accordance with the manufacturers’s NLRP1 Formulation protocol. All miRNA transfections had been for 72h. miR-3607 precursor (AM17100), negative manage (miR-CON) (AM17110), anti-miR-3607 inhibitor (MH19335), anti-miR-control inhibitor (4464076) had been applied for transfections.Mol Cancer Ther. Author manuscript; offered in PMC 2015 July 01.Saini et al.PageTissue samples and Ethics statementAuthor Manuscript Author Manuscript Author Manuscript Author ManuscriptFormalin-fixed, paraffin-embedded (FFPE) PCa samples were obtained from the SFVAMC. Written informed consent was obtained from all patients and also the study was approved by the UCSF Committee on Human Investigation (Approval quantity: H9058-35751-01). All slides were reviewed by a board certified pathologist for the identification of PCa foci too as adjacent standard glandular epithelium. RNA and miRNA extraction Total RNA was extracted from microdissected FFPE tissues applying a miRNeasy FFPE Kit (Qiagen) and an miRNeasy mini kit (Qiagen) was utilized for miRNA extraction from cultured cells following the manufacturer’s instructions. Migration, invasion and clonogenicity assays Cytoselect cell migration and invasion assay kit (Cell Biolabs, Inc.) was employed for migration and invasion assays, based on the manufacturer’s protocol. Briefly, 48 hrs posttransfection, cells have been counted and placed on handle inserts or Matrigel inserts at 1 ?05 cells/ml in serum-free medium and have been permitted to migrate for 20 h at 37 . Cells were removed from the prime of the inserts and cells that migrated/invaded though the polycarbonate/basement membrane had been fixed, stained and quantified at OD 560nm right after extraction. For clonogenicity assay, cells were counted, seeded at low density (1000 cells/ plate) and allowed to develop till visible colonies appeared. Then, cells had been stained with Giemsa and colonies had been counted. Cell viability assays Cell viability was determined at 24, 48, 72 hours by utilizing the CellTiter 96 AQueousOne Answer Cell Proliferation Assay Kit (Promega), in line with the manufacturer’s protocol. Flow Cytometry Fluorescence-activated cell-sorting (FACS) analysis was accomplished 72 hours post-transfection. The cells were harvested, washed with cold PBS, and resuspended in DAPI nuclear stain for cell cycle evaluation. Cells were staine.
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