Resveratrol-Induced Gene Expression Profiles in Human Prostate Cancer Cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE4299 | GEO | 2006/02/25
SECONDARY ACCESSION(S): PRJNA104661
REPOSITORIES: GEO
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Project description:Cell lines. HEK293 human embryonic kidney cells were obtained from ATCC (American Type Culture Collection, Manassas, VA) and were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Heat inactivated, Hyclone, Logan, Utah) and 1X antibiotics/glutamine (100 units/ml penicillin, 100ug/ml streptomycin, 292ug/ml L-Glutamine sulfate, all from Invitrogen Corp, Carlsbad, CA.), under either hypoxic (1% O2) or normoxic (21% O2) conditions at 37C in a tissue culture incubator. Treatment. Cells treated in hypoxia for 16 hours or transfected with HIF-1a (HIF-1amut or HIF2a) plasmid. At the end of treatment, cells were collected and washed. Microarray studies. Total RNA samples were extracted from HEK293T cells grown in 1% or 21% O2 overnight and from transfected cells. Total RNA (20 µg) from each sample was synthesized into double-stranded cDNA with reverse transcriptase (Fairplay labeling kit, Stratagen, La Jolla, CA) using an oligo d(T) primer. The double stranded cDNA from untreated cells was labeled with Cy3 monofunctional reactive dye and that from hypoxia-treated or transfected cells was labeled with Cy5 monofunctinal reactive dye. The probe was hybridized to a long oligo array (Hs-Operon V2-vB1.1px.gal) containing 20K human transcripts (NCI Microarray Facility, Advanced Technology Center, Gaithersburg, MD) overnight at 42C . The slides were washed and spin-dried. Microarray slides were scanned with a GenePix 4000 microarray scanner (Axon Instruments, Union City, CA.). The microarray images were analyzed with GenePix 3.0 software and data analysis was performed with the MicroArray Database (mAdb) system hosted by the Center for Information Technology and Center for Cancer Research at the NCI. Keywords: other
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Project description:Analysis of a panel of 41 human ovarian cancer cell lines for the study of ovarian cancer biology, drug response and drug sensitivity. Ovarian cancer cell lines were either obtained from the American Type Culture Collection (ATCC, Manassas, VA) (CAOV3, OV90, OVCAR3, SKOV3, TOV112D), the European Collection of Cell Cultures, Salisbury, England (A2780CP, A2780S); Kyoto University, Kyoto, Japan (CHI, CHIcisR, M41, M41CSR, Tyknu, and TyknuCisR), or were kind gifts from Dr. Patricia Kruk, Department of Pathology, College of Medicine, University of South Florida, Tampa, FL; and Susan Murphy, PhD, Dept of OBGYN/Division of GYN Oncology, Duke University, Durham, NC (A2008, C13, CAOV2, FUOV1, HeyA8, IGR-OV1, IMCC3, IMCC5, MCAS, OV2008, OVCA420, OVCA429, OVCA432, OVCA433, OVCAR4, OVCAR5, OVCAR8, Dov13, BG1, Ovary1847, OVCAR10, OVCAR2, SK-OV-4). Cell lines were maintained in RPMI-1640 (Invitrogen; Carlsbad, California) supplemented with 10% fetal bovine serum (Fisher Scientific; Pittsburg, PN), 1% sodium pyruvate, 1% penicillin/streptomycin (Cellgro; Manassas, VA), and 1% nonessential amino acids (HyClone; Hudson, New Hampshire). Mycoplasma testing was performed every six months following manufacturer’s protocol (Lonza, Rockland ME).
2012-06-30 | E-GEOD-34615 | biostudies-arrayexpress
Project description:MRC-5 cells (American Type Culture Collection, Manassas, VA) derived from human lung fibroblasts (passages 19-25) were cultured in MEM with Earle’s salts, supplemented with 2 mM glutamine and 10% heat-inactivated FCS. Cells were grown in an incubator with a humidified atmosphere of 5% CO2 in air at 37oC for several days to reach confluence. HCMV strain AD169 (American Type Culture Collection, Manassas, VA) was used in these studies. A continually replenished stock of HCMV was cultured in confluent MRC-5 cells. At the appropriate time, the infected cells were lysed and the infectivity of the resultant viral stock was assayed by plaque assay. For the studies reported here, HCMV infection was carried out by exposing the confluent MRC-5 cells to a virus stock at a multiplicity of infection (MOI) of ~3-5 plaque-forming units (PFU) per cell for 1 h at 370C. To control for non-specific effects of the cell lysate portion of the viral stock, a parallel set of MRC-5 cells were always mock infected by a 1h exposure to a lysate of uninfected MRC-5 cells. After the infectious period of exposure, both mock- and virus-treated cells were rinsed with phosphate buffered saline (PBS) followed by fresh culture medium and returned to cell culture for various post-exposure (PE) times. Mock- or HCMV-infected cells were homogenized in guanidinium isothiocyanate, followed by the isolation of total RNA following the method of Chirgwin et al. (PubMed ID 518835). Purity and integrity of the RNA were checked spectroscopically and by gel electrophoresis prior to use. Two successive oligo (dT) cellulose columns were then used to isolate poly A+ RNA for microarray analysis, yielding 600 ng of mRNA per channel (mock-infected or HCMV-infected cells). These were labeled with Cy3 and Cy5 dyes respectively prior to hybridization with a UniGEM V cDNA microarray (Incyte Genomics, St Louis, MO). Keywords: other
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Project description:Cytokines are implicated in the development of inflammatory diseases such as endometriosis. This project was designed to test the hypothesis that specific cytokines that are secreted by macrophages, such as tumor necrosis factor ? (TNF?) and interleukin 1 beta (IL1?), cause gene expression changes in endometrial stromal cells. Telomerase-immortalized human endometrial stromal cells (T-HESC) were treated with TNF? (5ng/ml) ± IL1? (1ng/ml). DNA microarray and real time RT-PCR were used to study the gene expression changes in T-HESC cells. Two hundred and nineteen genes featuring in various gene ontologies were found to be differentially expressed in T-HESC cells treated with TNF? ± ILI?. 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Keywords: cytokines, endometrial stromal cells, endometriosis Experimental factors: cytokine treatment Experimental design: The telomerase immortalized - human endometrial stromal cell line (T-HESC, ATCC #CRL-4003, Manassas, VA) was used for all experiments. The cells were treated with vehicle or with the cytokines, TNF? (5ng/ml) ± IL1? (1ng/ml). Three different passages of cells were collected for each treatment group to provide biological replicates. Quality control steps: The cRNA that was synthesized from each cell culture sample was used for hybridization to a single CodeLink (Applied Microarrays, Tempe, AZ) whole human genome microarray. Only one sample was hybridized with each slide and only one dye (Alexa 647) was used so no dye swaps were necessary. Bacterial control spikes were used as per manufacturer's instructions. 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Hybridization procedures and parameters: 10 micrograms of cRNA was mixed with fragmentation buffer and heated to 94°C for 20 min. The fragmented cRNA was mixed with CodeLink hybridization buffer, loaded on the microarray slides, and hybridized for 18 hours at 37°C. The slides were washed in 0.75x TNT (Tris-HCl, NaCl, Tween-20) at 46°C for 1h then incubated with streptavidin-Alexa 647 fluorescent dye for 30 min at room temperature. The Alexa fluor was prepared in TNB blocking buffer (0.1M Tris-HCl, 0.15M NaCL, 0.5% NEN Blocking Reagent-PerkinElmer) The slides were then washed 4 times for 5 min each in 1x TNT each and once in 0.05% Tween 20 for 20 sec. The slides were dried by centrifugation and scanned in an Axon GenePix 4000B scanner.
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