Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Transcription profiling of human HeLa cells in response to IKAP/hELP1 siRNA transfection and/or tumor necrosis factor-a treatment

ABSTRACT: HeLa cells were un-transfected or transfected with specific siRNAs (20µM) targeting IKAP/hELP1 or non-specific siRNAs against GFP. Same experiment was done by stimulating the cells for 1 hour with TNF-a (200U/ml). conditions: 1/untransfected 2/siRNAs IKAP 3/untransfected + TNF-a 200U/ml 1h 4/siRNAs IKAP + TNF-a 200U/ml 1h 5/siRNAs GFP + TNF-a 200U/ml 1h

ORGANISM(S): Homo sapiens

SUBMITTER: Marie-Paule Merville

PROVIDER: E-MEXP-641 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Transcription impairment and cell migration defects in elongator-depleted cells: implication for familial dysautonomia.

Close Pierre P Hawkes Nicola N Cornez Isabelle I Creppe Catherine C Lambert Charles A CA Rogister Bernard B Siebenlist Ulrich U Merville Marie-Paule MP Slaugenhaupt Susan A SA Bours Vincent V Svejstrup Jesper Q JQ Chariot Alain A

Molecular cell 20060501 4

Mutations in IKBKAP, encoding a subunit of Elongator, cause familial dysautonomia (FD), a severe neurodevelopmental disease with complex clinical characteristics. Elongator was previously linked not only with transcriptional elongation and histone acetylation but also with other cellular processes. Here, we used RNA interference (RNAi) and fibroblasts from FD patients to identify Elongator target genes and study the role of Elongator in transcription. Strikingly, whereas Elongator is recruited t ...[more]

PMID: 16713582

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Project 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, 25Â°C) and stringent (1 M NaCl, 50Â°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.

Dataset Information

Transcription profiling of human HeLa cells in response to IKAP/hELP1 siRNA transfection and/or tumor necrosis factor-a treatment

Publications

Transcription impairment and cell migration defects in elongator-depleted cells: implication for familial dysautonomia.

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