Differential Gene Up-regulation by HIF-1a and HIF-2a in HEK293T.
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE2020 | GEO | 2005/04/01
SECONDARY ACCESSION(S): PRJNA91843
REPOSITORIES: GEO
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2005-01-31 | GSE2098 | GEO
Project description:Description This series contains microarrays for tumor associated macrophages (TAMs) purified from 8 week old male C57/BL6N mice, 3 weeks after tumor implantation into the left hind limb. Peritoneal exudates cells (PECs) were also harvested from control C57/BL6N mice. The resulting adherent populations were maintained in culture and resting TAMs and PECs were treated for 4 hours with 100 ng/mL of LPS to promote macrophage activation. RNA isolation and cDNA microarrays: Total RNA was isolated from approximately 20x10e6 PEC and TAM cells using standard Trizol purification protocols (Invitrogen, Carlsbad, CA). Microarrays were manufactured at the NCI, Laboratory of Molecular Technology (Frederick MD). The 10,368 Mm UniGem2 set was printed on poly-lysine coated glass slides and hybridized according to NCI facility protocols. All slides were scanned using as Axon GenePix 4000 scanner (Axon Instruments, Foster city, CA) and resulting data was uploaded for analysis to the mAdb microarray database (http://madb.nci.nih.gov/). Keywords = tumor associated macrophage
2010-06-10 | E-GEOD-2098 | biostudies-arrayexpress
Project description:Cell lines. TK6 and WTK1 are EBV-immortalized human lymphoblastoid cell lines that have been derived from the same donor. TK6 cells bear wild-type TP53 tumor suppressor, while WTK1 cells bear a mutant TP53 containing a C-to-T transition in exon 7, resulting in a change of Met to Ile at codon 237, and coding for a functionally inactive protein. Cells were maintained in exponentially growing suspension culture at 37ºC in a humidified, 5% CO2 atmosphere in RPMI medium1640 supplemented with 10% heat-inactivated calf serum, 100 units/ml penicillin, 100 mg/ml streptomycin, and 2 mM L-glutamine. Stock cells were subcultured and maintained at a density not greater than 1.5 x 106 cells per ml in 150-mm dishes throughout experiments. NO treatment. Cells at a density of 4 x 105 cells per ml in 100 ml of custom RPMI 1640 medium without calcium nitrate (GIBCO) and calf serum were exposed to pure NO gas (Matheson, Gloucester, MA) by diffusion through Silastic tubing (0.025 in. ID, 0.047 in OD, Dow Corning, Midland, MI) delivery system. NO diffuses through this permeable membrane at a constant rate, and was delivered through 30 cm-long tubing into the medium of well-stirred cell suspensions for 2 h. Cells similarly exposed to argon gas served as negative controls. Total dose (and rate) of NO delivered under these conditions was 390 mmol (533 nM/s), calculated from nitrite plus nitrate content of the medium as determined by automated analysis using the Griess reagent [N-(1-napthy)-ethylenediamine and sulfanilic acid]. At the end of treatment, cells were collected by centrifugation, washed once, resuspended in fresh culture medium containing 10% heat inactivated calf serum, and incubated at 37 ºC. At the indicated times, cells were washed in cold phosphate-buffered saline, harvested and stored at -80 ºC for RNA extraction. cDNA microarray hybridization and analysis. Cells were lysed with Trizol reagent (INVITROGEN, Carlsbad, CA), and total RNA was extracted according to the manufacturer's instructions at 0, 6, 12, and 24h after NO treatment. Fluorescently-labeled cDNA probes were generated using 40 µg of total RNA by a single round of reverse transcription in the presence of aminoallyl-dUTP (SIGMA, St. Louis, MO), followed by a coupling reaction to Cy3 or Cy5 monofunctional NHS-ester (Amersham Pharmacia, Piscataway, NJ). Complex probes (containing untreated (time 0) Cy3-labeled cDNA and NO or argon-treated Cy5-labeled cDNA) were denatured and hybridized to glass slides featuring 9, 180 cDNA clones based on the Hs Unigene build #131 platform (GPL 257), and printed by the NCI Microarray Facility, Advanced Technology Center, Gaithersburg, MD. Upon overnight incubation at 42 ºC, the slides were washed successively in 1 X SSC/ 0.1% SDS, 1 X SSC and 0.2 X SSC for 2 min each, then rinsed in 0.5 X SSC and spin-dried. The two fluorescent intensities were measured simultaneously using a GenePix 4000A scanner, and the acquired image was processed with GenePix Pro 3.0 software (Axon Instruments, Union City, CA). Keywords: time-course
2004-04-01 | GSE1064 | GEO
Project description:A comparison of gene expression between control versus IPF human lung MPC using human Affy 1.0st chips. This work was funded by grants to S.M. Majka from the NIH R01HL091105 and NIH R01HL11659701. Additional funding was also provided by PPG-5P01HL108800-04 (PI:J. Loyd). Experiments were performed using the University of Colorado Cancer Center Microarray core (NCI P30 CA 46934-14). The project was supported in part by the National Center for Research Resources, Grant UL1 RR024975-01, and is now at the National Center for Advancing Translational Sciences, Grant 2 UL1 TR000445-06.
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Project description:Human umbilical vein endothelial cells (HUVEC) were cultivated on 1%-gelatin-coated 75-cm2 culture flasks in Medium 200 supplemented with 2% fetal calf serum, penicillin (100 units/ml), streptomycin (100 units/ml) and Funizone (0.25 μg/ml) supplied by Cascade Biologics (Portland, OR). Prior to an experiment, HUVECs were subcultivated with Trypsin/EDTA onto 1% gelatin coated glass 30-mm glass coverslips in 60 mm glass Petri dishes. The hypoxic treatment was performed in airtight chambers from Mitsubishi Gas Chemical Co. Inc (Remel Scientific, Baton Rouge, LA) Preconditioning consisted of 1-hour hypoxia followed by various periods of reoxygenation (0, 3, 5, 12 and 24 hrs) for gene expression analysis. Total RNA was extracted from cultured HUVECs with TRITM reagent according to the manufacture’s instructions (Molecular Research Center, Cincinnati, OH). Cy5 and Cy3 labeled cDNA probes were generated from total RNA by reverse transcription in the presence of aminoallyl-dUTP according to the method described by Xiang, et al,15. Human universal reference RNA (Stratagene, La Jolla, CA) was Cy3-labeled and used as reference RNA and RNA isolated from HUVEC at 0, 3, 5, 12 and 24 hour following reoxygenation were Cy5-labeled. The high-resolution scans of hybridized microarrays were acquired with a GenePix scanner 4000B (Axon Instruments, Inc., Union City, CA) and tabulated with GenePix pro 5.1 software. Keywords: time-course
2004-02-18 | GSE1041 | GEO
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2003-11-25 | GSE779 | GEO
Project description:Cells: ACH-2, and A3.01, were obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH. ACH-2, is a chronically infected cell line harboring HIV-1 LAV strain, while A3.01 is the corresponding parental uninfected cell line. Cells were grown in RPMI-1640 (Invitrogen, San Diego, CA) with 10% fetal bovine serum (FBS, Invitrogen), 5% penicillin-streptomycin (Invitrogen), and 2mM glutamine (Invitrogen). Cells were maintained at a concentration of 1x10e6 cells/ml in T-175 flasks. Cell concentrations and cell viability were monitored throughout the experiment at all time points studied. Cells were induced by addition of 20 ng/mL of phorbol myristyl acetate (PMA or TPA, Sigma, St Louis, MO) for one hour, after which the cells were washed with phosphate buffered saline (PBS). Cells were harvested by centrifugation at 1000 rpm for 10 minutes, at the following times after induction, 0.5, 3, 6, 8, 12, 18, 24, 48, 72, and 96 hour(s). HIV-infected and uninfected cells maintained and harvested in parallel with the PMA-treated cells, but not induced with PMA ( No PMA)were also harvested. Harvested cells were washed thrice with ice-cold PBS to remove media; cell pellets were snap frozen using ethanol-dry ice mixture and stored at (80°C for subsequent RNA extraction. Total RNA Extraction: Total RNA was extracted using RNEasy Midiprep Kits per manufacturer's protocol (Qiagen, Valencia, CA). RNA concentrations and purity were measured by spectrophotometry, RNA quality (absence of RNA degradation) was assessed by gel electrophoresis. RNA concentration was adjusted to the levels required for subsequent microarray experiment protocols by concentration in a SpeedVac (Savant Instruments, Holbrook, CA). RNA samples (6-7 µg/µL) were stored in 100 µL TE buffer at -80°C. Microarray Studies: Total RNA obtained from induced chronically infected and corresponding uninfected parental cells were used for microarray experiments. For each time point, RNA from the induced chronically infected ACH-2 cells and RNA from the corresponding induced, uninfected A3.01 cells were compared to minimize effects due to PMA induction. Microarrays were obtained from the National Cancer Institute Microarray Facility, Advanced Technology Center (Gaithersburg, MD). The microarrays (Hs. UniGem2) contained 10,368 cDNA spots on each glass slide. The cDNAs were selected for spotting on the slides based on their known or probable involvement in oncogenesis, signal transduction, apoptosis, immune function, inflammatory pathways, cellular transport, transcription, protein translation and other important cellular functions. A number of expressed sequence tags (ESTs) from unknown genes homologous to known genes and cDNAs encoding housekeeping genes were also included in these gene sets. For each time point, 50 µg of total RNA from PMA induced ACH-2 cells and 70 µg of total RNA from PMA induced A3.01 cells was labeled with Cy-3-dUTP and Cy-5-dUTP respectively. Higher amounts of RNA were used for Cy-5 labeling to minimize the disparities in dye incorporation. Each sample of RNA from PMA-induced, infected cells from a particular time point was compared with RNA from the corresponding PMA-induced, uninfected cells from the same time point for subsequent hybridization to the same array to ensure accurate comparisons and to eliminate inter-array variability. Following reverse transcription, the labeled cDNAs were then combined and purified using MicroCon YM-30 (Millipore, Bedford, MA) spin column filters, to remove any unincorporated nucleotides. 8-10 µg each of Cot-1 DNA, (Boehringer Mannheim, Indianapolis, IN), yeast tRNA (Sigma) and polyA (Amersham Biosciences) were added to the reaction mixture and heated at 100°C for 1 minute. Hybridization of the labeled cDNA to the microarray was carried out at 65°C overnight, followed by washes with 1X SSC, 0.2X SSC and 0.05X SSC respectively. The slides were dried by centrifugation at 1000 rpm for 3 minutes and then scanned as described below. Microarray Scanning and Data Analysis: The slides were scanned using an Axon GenePix 4000 scanner (Axon Instruments, Union City, CA). The photomultiplier tube values (PMT) were adjusted to obtain equivalent intensities at both wavelengths used, 635 nm and 532 nm for the Cy5 and Cy3 channels respectively. Image analysis was performed using GenePix analysis software (Axon Instruments) and data analysis was performed using the microArray Database (mAdb) system hosted by the Center for Information Technology and Center for Cancer Research at NIH (http://nciarray.nci.nih.gov/). Keywords = HIV Keywords = latency Keywords: time-course
2004-08-16 | GSE1441 | GEO
Project description:Cells: ACH-2, A3.01, J1.1, and U1 cells were obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH. U-937 cells were obtained from American Type Culture Collection (Manassas, VA). ACH-2, J1.1 and U1 are chronically infected cell lines harboring HIV-1 LAV strain, while A3.01, Jurkat, and U-937 are the corresponding parental uninfected cell lines. Cells were grown in RPMI-1640 (Invitrogen, San Diego, CA) with 10% fetal bovine serum (FBS, Invitrogen), 5% penicillin-streptomycin (Invitrogen), and 2mM glutamine (Invitrogen). Cells were maintained at a concentration of 1x10e6 cells/ml in T-175 flasks. Cell concentrations and cell viability were monitored throughout the experiment. Cells were harvested by centrifugation at 1000 rpm for 10 minutes. Harvested cells were washed thrice with ice-cold PBS to remove media; cell pellets were snap frozen using ethanol-dry ice mixture and stored at -80°C for subsequent RNA extraction. In order to compare expression profiles of chronically infected cell lines, ACH-2, U1, and J1.1 and their uninfected parental cell lines, A3.01, U-937 and Jurkat cells respectively, were grown under identical conditions but in presence of AZT (250 nM) in growth media and cells were harvested as described above. In these studies, no inducing agent was used in either chronically infected or uninfected parental cell lines so as to study changes in cellular gene expression cells latently infected with HIV and uninfected cells. Total RNA Extraction: Total RNA was extracted using RNEasy Midiprep Kits per manufacturer's protocol (Qiagen, Valencia, CA). RNA concentrations and purity were measured by spectrophotometry, RNA quality (absence of RNA degradation) was assessed by gel electrophoresis. RNA concentration was adjusted to the levels required for subsequent microarray experiment protocols by concentration in a SpeedVac (Savant Instruments, Holbrook, CA). RNA samples (6-7 µg/µL) were stored in 100 µL TE buffer at -80°C. Microarray Studies: Total RNA obtained from chronically infected and corresponding uninfected parental cells were used for microarray experiments. For each cell line, RNA from the chronically infected cells (ACH-2, J1.1 or U1)and RNA from the corresponding induced, uninfected cells (A3.01 , Jurkat or U937) were compared on the same array. Microarrays were obtained from the National Cancer Institute Microarray Facility, Advanced Technology Center (Gaithersburg, MD). The microarrays (Hs. UniGem2) contained 10,368 cDNA spots on each glass slide. The cDNAs were selected for spotting on the slides based on their known or probable involvement in oncogenesis, signal transduction, apoptosis, immune function, inflammatory pathways, cellular transport, transcription, protein translation and other important cellular functions. A number of expressed sequence tags (ESTs) from unknown genes homologous to known genes and cDNAs encoding housekeeping genes were also included in these gene sets. For each array, 50 µg of total RNA from chronically infected cells and 70 µg of total RNA from corresponding uninfected cells was labeled with Cy-3-dUTP and Cy-5-dUTP respectively. Higher amounts of RNA were used for Cy-5 labeling to minimize the disparities in dye incorporation. Following reverse transcription, the labeled cDNAs were then combined and purified using MicroCon YM-30 (Millipore, Bedford, MA) spin column filters, to remove any unincorporated nucleotides. 8-10 µg each of Cot-1 DNA, (Boehringer Mannheim, Indianapolis, IN), yeast tRNA (Sigma) and polyA (Amersham Biosciences) were added to the reaction mixture and heated at 100°C for 1 minute. Hybridization of the labeled cDNA to the microarray was carried out at 65°C overnight, followed by washes with 1X SSC, 0.2X SSC and 0.05X SSC respectively. The slides were dried by centrifugation at 1000 rpm for 3 minutes and then scanned as described below. Microarray Scanning and Data Analysis: The slides were scanned using an Axon GenePix 4000 scanner (Axon Instruments, Union City, CA). The photomultiplier tube values (PMT) were adjusted to obtain equivalent intensities at both wavelengths used, 635 nm and 532 nm for the Cy5 and Cy3 channels respectively. Image analysis was performed using GenePix analysis software (Axon Instruments) and data analysis was performed using the microArray Database (mAdb) system hosted by the Center for Information Technology and Center for Cancer Research at NIH (http://nciarray.nci.nih.gov/). Keywords = HIV Keywords = Latency Keywords: other
2004-08-16 | GSE1443 | GEO
Project description:In this study we used expression microarrays as an entry point to gain further insights into the differences between endocycling cells and mitotic cycling in tissues of the Drosophila larva. Gene expression analyses were performed on tissues hand dissected from yw67c2 early 3rd instar larval tissues collected 70-75hr after egg laying. Total RNA was isolated using RNeasy Micro Kit (QIAGEN Valencia, CA 91355), and reverse transcribed and fluorescently labeled with Cy3 or Cy5 (Amersham) using Amino Allyl MessageAmp™ II aRNA Amplification Kit (Ambion, Austin, TX 78744), according to the manufacturer's instructions. This was used to probe the DGRC-2 array, a Drosophila oligonucleotide array created by the Drosophila Genomics Resource Center (DGRC) which represents ~93% of annotated Drosophila genes from genome release 4.1 (Bogart et al., 2006). Two color microarray hybridizations were repeated with material from independent dissections representing two (fat body versus brain) and three (salivary gland versus brain) independent biological replicates. Slides were scanned by GenePix 4100A (Axon Instruments, Union City, CA) and the data was extracted with Axon GenePix Pro 5 image analysis software.
2010-08-12 | E-GEOD-19029 | biostudies-arrayexpress
Project description:Analysis of four lung cancer cell lines transfected with a vector expressing the transcriptional repressor Snail versus a vector control. Aberrant Snail expression is known to induce an EMT program in lung cancers. Four lung cancer cell lines (H292, H358, H441, H1437) with stable overexpression of the human SNAI1 gene and vector expressing controls were collected at equal confluency with the miRNeasy Mini kit (Qiagen). One microgram of total RNA was labeled using miRCURY LNA™ microRNA Array Power Labeling kit by the UCLA Clinical Microarray Core. The labeled miRNAs were hybridized to Exiqon miRCURY LNA microRNA Array-6th Generation according to the manufacturer’s instructions. This array includes 927/648/351 human/mouse/rat miRNAs as well as 438 miRPlus miRNAs. The miRNA array slides were scanned using Axon GenePix 4000B scanner (Axon Instruments, Foster City, CA) and processed by using the GenePix Pro 6.0 software (Axon Instruments). The raw data were normalized by using a combination of housekeeping miRNA, spike-in miRNA and invariant endogenous miRNAs.
2013-07-17 | E-GEOD-48922 | biostudies-arrayexpress