Resveratrol-Induced Gene Expression Profiles in Human Prostate Cancer Cells
ABSTRACT: Cell Culture and Treatments The LNCaP cell line was obtained from American Type Culture Collection (Manassas, VA) and maintained in RPMI medium with 10% fetal bovine serum (CDT; Hyclone Laboratories, Logan, UT) and penicillin/streptomycin (Mediatech, Herndon, VA) in an environment of 95% air and 5% CO2 at 370C. Upon reaching 75% confluence, cells were treated with either DMSO control or purified resveratrol at several concentrations (LKT Laboratories, St. Paul, MN), dissolved in DMSO, and incubated for varying lengths of time. Final concentration of DMSO in media was 0.01%. Total RNA was prepared from cells using TRIzol (Invitrogen Life Technologies, Carlsbad, CA). An all pairs experiment design type is where all labeled extracts are compared to every other labeled extract. Keywords: all_pairs Overall design: Computed
Project description:Cell Culture and Treatments The LNCaP cell line was obtained from American Type Culture Collection (Manassas, VA) and maintained in RPMI medium with 10% fetal bovine serum (CDT; Hyclone Laboratories, Logan, UT) and penicillin/streptomycin (Mediatech, Herndon, VA) in an environment of 95% air and 5% CO2 at 370C. Upon reaching 75% confluence, cells were treated with either DMSO control or purified resveratrol at several concentrations (LKT Laboratories, St. Paul, MN), dissolved in DMSO, and incubated for varying lengths of time. Final concentration of DMSO in media was 0.01%. Total RNA was prepared from cells using TRIzol (Invitrogen Life Technologies, Carlsbad, CA). An all pairs experiment design type is where all labeled extracts are compared to every other labeled extract. Computed
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).
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
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
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 α (TNFα) and interleukin 1 beta (IL1β), cause gene expression changes in endometrial stromal cells. Telomerase-immortalized human endometrial stromal cells (T-HESC) were treated with TNFα (5ng/ml) ± IL1β (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 TNFα ± ILIβ. The gene ontologies included functions expected to be associated with the development of endometriosis such as peptidases, cell adhesion, cell death/apoptosis, cell cycle, growth factors, cytoskeletal organization, defense/immune system, signal transduction, and transcriptional regulation. The differential expression of 4 genes (interleukin 8 (IL8), interleukin 6 (IL6), IL1β and matrix metalloproteinase (MMP3) was confirmed by real time RT-PCR. All four genes were up-regulated in response to TNFα ± ILIβ in T-HESC cells. The effect of TNFα ± ILIβ on migration and invasion of T-HESC cells, as measured with Boyden chambers, was not affected by treatment with these cytokines. Goal of the experiment: To examine differential gene expression in telomerase-immortalized human endometrial stromal cells (T-HESC) in response to treatment with tumor necrosis factor alpha (TNFα) ± interleukin 1β (IL1β). 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, TNFα (5ng/ml) ± IL1β (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. Samples used, extract preparation and labeling: The origin of each biological sample: The samples were cultured cells of the telomerase-immortalized human endometrial stromal cell (T-HESC) line obtained from ATCC (CRL-4003). Manipulations of biological samples and protocols used: The telomerase immortalized - human endometrial stromal cell line (T-HESC, ATCC #CRL-4003, Manassas, VA) was used for all experiments. The cells were routinely maintained in Dulbecco’s modified Eagle’s medium (DMEM, Sigma, St. Louis, MO) with 10% fetal bovine serum albumin (FBS, Hyclone, South Logan, UT), insulin, selenious acid, transferrin, bovine serum albumin and linoleic acid (ITS+ Premix, BD Biosciences, Bedford, MA), puromycin (Cellgro, Manassas, VA) and penicillin/ streptomycin (Hyclone, South Logan, UT). After the cells reached 80% confluence they were starved for 24 hours in T-HESC starvation medium (DMEM+ ITS+ puromycin+ penicillin/streptomycin) before treatment. The cells were treated with vehicle or with TNFα (5ng/ml) alone, IL1β (1ng/ml) alone, or with the two cytokines combined at the same concentrations for 48 hours. Total RNA was then isolated from the cell cultures for use in the DNA microarray experiments. Each treatment was carried out with 3 different passages of cells. Technical protocols: The cultured endometrial cells were rinsed with PBS and TRI reagent (MRC) was added. The cells were scraped into tubes and centrifuged in the presence of bromochloropropane and sodium acetate. The RNA-containing layer was removed and the RNA purified on an RNeasy extraction column (Qiagen). The sample was treated with an on-column DNase treatment (RNase-free DNase, Qiagen). The purity and quantity was evaluated with the RNA 6000 NanoLabChip in an Agilent Bioanalyzer. Labeled cRNA was prepared using the MessageAmp II-Biotin enhanced kit (Ambion). 1.0 microgram of total T-HESC cell RNA was mixed with bacterial control RNA spikes and primed with T7 oligo(dT) primer for 10 min at 70C. (The bacterial control spikes included araB, entF, fixB, gnd, hisB, and leuB.) The first strand of cDNA was synthesized with first strand buffer, dNTP mix, RNase inhibitor, and reverse transcriptase for 2 h at 42C. The second strand cDNA synthesis reaction was prepared using second strand buffer, dNTP mix, DNA polymerase mix, and RNase H; the reaction was carried out for 2h at 16C. The double-stranded cDNA was purified on columns. The double-stranded cDNA was mixed with T7 reaction buffer, T7 ATP, T7 GTP, T7 UTP, T7 CTP, biotin-11-UTP, and T7 enzyme mix for the synthesis of cRNA. The cRNA synthesis reaction was terminated after 14h at 37C by purifying the cRNA on columns. The concentration of cRNA was determined by spectrophotometry. 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.
Project description:Tamoxifen is the most widely administered adjuvant first-line hormone therapy for Estrogen receptor α (ERα) positive breast cancer patients. However, one from three patients will develop resistance, while the underlying molecular mechanisms are currently unclear. Recent studies reported that abnormal expression of miRNAs played a role in cancer progress. To study the potential function of miRNAs in tamoxifen resistance, Affymetrix GeneChip® miRNA 3.0 microarray was employed to identify differentially expressed miRNAs between tamoxifen sensitive MCF7 parent (MCF7-Pa) cells and induced resistant (MCF7-Re) cells. Overall design: The human breast cancer MCF7 cell line was obtained from the American Type Culture Collection (Manassas, VA, USA). MCF7 parent (MCF7-Pa) cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 40U/ml insulin. MCF7-derived tamoxifen resistant sublines (MCF7-Re) were developed by culturing in 1640 medium without phenol red (Life Technologies, USA) supplemented with 5% charcoal-stripped FBS (cFBS) (HyClone, USA) and 1 uM 4-hydroxytamoxifen (Sigma-Aldrich, MO, USA) for 1 year. After cultured in RPMI 1640 medium without phenol red supplemented with 5% cFBS and without tamoxifen for 7 days, MCF7-Pa and MCF7-Re cells total RNA was harvested using TRIzol® reagent (Life Technologies, USA) according to the manufacturer’s instructions. RNA extraction and miRNA profile detection was provided by CapitalBio Corporation (Beijing, China) as generated as the manufacturer's recommendations (Affymetrix).
Project description:Cell Lines, Cultures and HCC Tumor Tissues. <br>Malignant and non-malignant hepatocyte cell lines were obtained from the American Type Culture Collection (Manassas, VA) and cultured as recommended by the supplier. Total RNA from three paires of normal human liver tissue and from hepatocellular carcinoma was obtained from BioChain Institute, Inc. (Hayward, CA).<br><br>Isolation of MicroRNA. <br>Total RNA was obtained from cell lines and tissue samples using the Totally RNA isolation kit (Ambion, Austin, TX). The miRNA fraction was obtained by flashPAGE purification using the flashPAGE Fractionator System (Ambion, Austin, TX). The size of the microRNA fractions were confirmed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Palo Alto, CA).
Project description:To identify the miRNA expressing profiles of Platelet microparticles（PMPs, we have employed the Agilent Human miRNA 8×60K (Design ID:046064) microarray. Platelet microparticles. The platelets were derived from citrated blood of healthy human donors under an Institutional Review Board-approved protocol. Platelets were isolated after centrifugation of blood (1200r for 30 min at 21℃), then the supernatant (platelet-rich plasma) was centrifuged at 2000r for 30 min at 21℃, and the pellet containing platelets was resuspended in RPMI-1640 medium (HyClone, Logan, UT). Platelets were counted (Clinical Laboratory, Shanghai First Maternity and Infant Hospital, Shanghai) and adjusted to a density of 150 × 106 cells/mL before supplement with 1.5% ACD(sigma ) and stimulated with thrombin (1.0 u/mL; Takeda Austria) for 1 h. PMPs were in the supernatant after centrifugation at 4000r for 10 min at 4℃,then the supernatants were centrifuged at 50,000 × g for 60 min at 4 °C. The pellets containing MPs were resuspended in RPMI-1640 medium and quantified by BCA method. The gene expressions of three independent paired PMPs from platelets stimulated by thrombin or apoptosis.
Project description:C2C12 mouse myoblasts (American Type Culture Collection, Manassas, VA) were maintained in growth medium (DMEM supplemented with 10% Fetal Bovine serum, Invitrogen Corporation, Carlsbad, CA) in 5% CO2. Cells were plated at a density of 40-50% in 100mm dishes. When cells reached ~90% confluency 24 hours later, they were switched to differentiation media (DMEM containing 2% horse serum; GIBCO, Invitrogen Corporation, Carlsbad, CA) which was subsequently changed every 48 hours. All cells were allowed to differentiate for 4 days with feeding every two days (day 0, 2, and 4). Beginning at day 4, control cells were re-fed every 48 hours whereas experimental cells were not re-fed. After an additional 4 days (8 days post differentiation), cells were harvested and total RNA was isolated from control and starved (non re-fed cells)