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Title: Transcription profiling of human tetracycline-regulated cell line expressing an NF-kB inhibitor to systematically identify NF-kB dependent genes      
dateReleased:
09-07-2007
description:
TNF time course series of HelA tet off cells cultured in presence or absence of Dox; TNF is a pro-inflammatory cytokine that controls expression of inflammatory genetic networks. Although the Nuclear Factor-kB (NF-kB) pathway is crucial for mediating cellular TNF responses, the complete spectrum of NF-kB dependent genes is unknown. In this study, we used a tetracycline-regulated cell line expressing an NF-kB inhibitor to systematically identify NF-kB dependent genes. A microarray data set generated from a time course of TNF stimulation in the presence or absence of NF-kB signaling was analyzed. Methods: Cell culture, treatment and transfection-The human cervical epithelioid carcinoma cell line HeLa expressing tTA (HeLa Tet-Off) pBI-EGFP- IkBa Mut was constructed earlier (23). HeLa Tet-Off were grown in medium containing 90 % Dulbecco's Modified Eagle's Medium (DMEM), 10 % heat-inactivated fetal bovine serum (FBS), 4mM L-glutamine, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 100 mg/ml G418, 100 units /ml penicillin G sodium, and 100 mg/ml streptomycin sulfate in a humidified atmosphere of 5 % CO2. For TNF stimulation, freshly isolated cells were split into two cultures, one group maintained in Dox, the other without for 7 d, a time at which IkBa Mut expression was maximal. Thereafter, recombinant human (rh) TNFa (25 ng/ml, final concentration) was added directly to the culture medium for indicated times prior to harvest. Oligonucleotide probe based microarray-Hu95Av2 GeneChip (Affymetrix Inc, Santa Clara, CA) containing 12,626 sequenced human genes were hybridized according to the manufacturer's recommendations and washed using both non-stringent (1 M NaCl, 25°C) and stringent (1 M NaCl, 50°C) conditions prior to staining with phycoerythrin streptavidin (10 mg/ml final). Arrays were scanned using a Gene Array Scanner (Hewlett Packard) and analyzed using the Gene Chip Analysis Suite 5 software (Affymetrix Inc). For each gene, 16-20 probe pairs are immobilized as ~25-mer oligonucleotides that hybridize throughout the mRNA; each probe pair is represented as a perfect match (PM) oligonucleotide and a mismatch (MM) oligonucleotide as hybridization control. The the Absolute Call [e.g., the gene is detected (present) or not (absent)] and the Signal Intensity (measure of mRNA abundance) is determined. Four independent experiments were performed identically including control (0 h), 1, 3 and 6 h TNF stimulation (25 ng/ml) in the presence or absence of Doxycyline (2 mg/ml) in growth medium. These are indicated as 180, 181, 220 and 266 series, and are considered replicates of each other.
privacy:
not applicable
aggregation:
instance of dataset
ID:
E-GEOD-2624
refinement:
raw
alternateIdentifiers:
2624
dateSubmitted:
05-04-2005
keywords:
functional genomics
dateModified:
05-02-2014
creators:
Allan Brasier
availability:
available
types:
gene expression
name:
Homo sapiens
ID:
A-AFFY-1
name:
Affymetrix GeneChip Human Genome U95Av2 [HG_U95Av2]
accessURL: https://www.ebi.ac.uk/arrayexpress/files/E-GEOD-2624/E-GEOD-2624.raw.1.zip
storedIn:
ArrayExpress
qualifier:
gzip compressed
format:
TXT
accessType:
download
authentication:
none
authorization:
none
accessURL: https://www.ebi.ac.uk/arrayexpress/files/E-GEOD-2624/E-GEOD-2624.processed.1.zip
storedIn:
ArrayExpress
qualifier:
gzip compressed
format:
TXT
accessType:
download
authentication:
none
authorization:
none
accessURL: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE2624
storedIn:
Gene Expression Omnibus
qualifier:
not compressed
format:
HTML
accessType:
landing page
primary:
true
authentication:
none
authorization:
none
abbreviation:
EBI
homePage: http://www.ebi.ac.uk/
ID:
SCR:004727
name:
European Bioinformatics Institute
homePage: https://www.ebi.ac.uk/arrayexpress/
ID:
SCR:002964
name:
ArrayExpress
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