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Transcription profiling by array of human MCF-7 breast cell clones expressing ER-beta tagged with TAP-tag at the C-terminal or N-terminal

ABSTRACT: MCF-7 TET Off cells (MCF-7 wt) were used to produce stable clones expressing ER-beta tagged with TAP-tag respectively at the C-term and at the N-term (C-TAP-ER-beta and N-TAP-ER-beta) or expressing ER-alpha tagged (C-TAP-ER-alpha) as previously described.Cells were cultured in standard growth conditions, then were lysed and RNA was extracted using TRIzol method.Total RNA were fluorescently labelled, amplified and hybridized in triplicate (MCF7-TAP and C-TAP-ER-alpha) and in duplicate (C-TAP-ER-beta_A, C-TAP-ER-beta_B, N-TAP-ER-beta) for 18 hours on Illumina v2 MicroRNA Expression BeadChips and after scanning, analysis was performed with GenomeStudio v.2010.1 software, for quality control and miRNA expression analysis.

ORGANISM(S): Homo sapiens

SUBMITTER: Weisz Alessandro

PROVIDER: S-ECPF-TABM-1053 | biostudies-other |

REPOSITORIES: biostudies-other

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Transcription profiling by array of human MCF-7 cells with ER-beta tagged with TAP-tag at the C-term or N-term or expressing ER-alpha tagged

Project description:MCF-7 TET Off cells (MCF-7 wt) were used to produce stable clones expressing ER-beta tagged with TAP-tag respectively at the C-term and at the N-term (C-TAP-ER-beta and N-TAP-ER-beta) or expressing ER-alpha tagged (C-TAP-ER-alpha). All were grown in Dulbecco's modified Eagle's medium (DMEM). Then cells were lysed and RNA extracted were pooled. For mRNA expression profiling, 500 ng total RNA were reverse transcribed and used for synthesis of cDNA and biotinylated cRNA. Finally cRNA were hybridized for 18 hours on Illumina HumanHT-12 v3.0 BeadChips and after scanning, data analysis was performed.

| S-ECPF-TABM-1052 | biostudies-other

Transcription profiling by array of human MCF-7 breast cell clones expressing ER-beta tagged with TAP-tag at the C-terminal or N-terminal

Project description:MCF-7 TET Off cells (MCF-7 wt) were used to produce stable clones expressing ER-beta tagged with TAP-tag respectively at the C-term and at the N-term (C-TAP-ER-beta and N-TAP-ER-beta) or expressing ER-alpha tagged (C-TAP-ER-alpha) as previously described.Cells were cultured in standard growth conditions, then were lysed and RNA was extracted using TRIzol method.Total RNA were fluorescently labelled, amplified and hybridized in triplicate (MCF7-TAP and C-TAP-ER-alpha) and in duplicate (C-TAP-ER-beta_A, C-TAP-ER-beta_B, N-TAP-ER-beta) for 18 hours on Illumina v2 MicroRNA Expression BeadChips and after scanning, analysis was performed with GenomeStudio v.2010.1 software, for quality control and miRNA expression analysis.

2011-10-11 | E-TABM-1053 | biostudies-arrayexpress

Transcription profiling by array of human breast cancer cell line MCF7 expressing TAP tagged ER-alpha following hormone treatment

Project description:MCF-7 TET Off cells (MCF-7 wt) were used to produce stable clones expressing ER-alpha tagged with TAP-tag at the C-term (C-TAP-ER-alpha). All cells were grown in Dulbecco's modified Eagle's medium (DMEM), starved by using DMEM w/o phenol red and 5% Dextran Coated Charcoal stripped serum (DCC-FBS) for 5 days. Cells were stimulated with 17 beta Estradiol (E2), 4-idrossi-tamoxifen (Tam), raloxifene (Ral) and Fulvestrant (ICI) at the concentration of 10-8M for 12 hours or with vehicle Ethanol. Biological replicate were lysed and RNA extracted were pooled. For mRNA expression profiling, 500 ng total RNA were reverse transcribed and used for synthesis of cDNA and biotinylated cRNA. Finally cRNA were hybridized for 19 hours on Illumina HumanHT-12 v4.0 BeadChips and after scanning, data analysis was performed.

| S-ECPF-MTAB-1196 | biostudies-other

Transcription profiling by array of human MCF-7 cells with ER-beta tagged with TAP-tag at the C-term or N-term or expressing ER-alpha tagged

2011-10-11 | E-TABM-1052 | biostudies-arrayexpress

MicroRNA profiling by array of human MCF-7 breast cancer cells with ER-beta tagged at the C-terminal or N-terminal and treated with 17-beta-estradiol

Project description:Human estrogen-responsive breast cancer cell line MCF-7 TET Off (MCF-7 wt) were used to produce stable clones expressing ER-beta tagged with TAP-tag respectively at the C-term and at the N-term (C-TAP-ER-beta and N-TAP-ER-beta) or expressing ER-alpha tagged (C-TAP-ER-alpha) as previously described.(C-TAP-ER-beta, N-TAP-ER-beta, C-TAP-ER-alpha) were treated with 10nM of17-beta-estradiol, or vehicle (ETOH). miRNA expression was analyzed on total RNA extracted before or after 6, 12, 24, and 72 hours hormonal stimulation. Total RNA was fluorescently labelled, amplified in triplicate, to be, than, pooled for the Hybridization.Each pool, were hybridized for 18 hours on Illumina v2 MicroRNA Expression BeadChips, and after scanning, analysis was performed with GenomeStudio v.2010.1 software, for quality control and miRNA expression analysis.

2011-07-29 | E-TABM-1055 | biostudies-arrayexpress

Transcription profiling by array of human MCF-7 breast cancer cells treated with tamoxifen or adenovirus expressing ERbeta

Project description:The beneficial effect of the selective estrogen receptor (ER) modulator tamoxifen in the treatment and prevention of breast cancer is assumed to be through its ability to antagonize the stimulatory actions of estrogen, although tamoxifen can also have some estrogen-like agonist effects. Here, we report that, in addition to these mixed agonist/antagonist actions, tamoxifen can also selectively regulate a unique set of >60 genes, which are minimally regulated by estradiol (E2) or raloxifene in ERalpha-positive MCF-7 human breast cancer cells. This gene regulation by tamoxifen is mediated by ERalpha and reversed by E2 or ICI 182,780. Introduction of ERbeta into MCF-7 cells reverses tamoxifen action on approximately 75% of these genes. To examine whether these genes might serve as markers of tamoxifen sensitivity and/or the development of resistance, their expression level was examined in breast cancers of women who had received adjuvant therapy with tamoxifen. High expression of two of the tamoxifen-stimulated genes, YWHAZ/14-3-3z and LOC441453, was found to correlate significantly with disease recurrence following tamoxifen treatment in women with ER-positive cancers and hence seem to be markers of a poor prognosis. Our data indicate a new dimension in tamoxifen action, involving gene expression regulation that is tamoxifen preferential, and identify genes that might serve as markers of tumor responsiveness or resistance to tamoxifen therapy. This may have a potential effect on the choice of tamoxifen versus aromatase inhibitors as adjuvant endocrine therapy.

| S-ECPF-GEOD-4025 | biostudies-other

Chip-chip from C. glutamicum wild type expressing GlxR tagged with Strep Tag II and cyaB deletion strain expressing GlxR tagged with Strep Tag II.

Project description:Corynebacterium glutamicum GlxR is a homolog of the cAMP receptor protein. Although over 200 GlxR binding sites in the C. glutamicum genome are predicted in silico, studies on the GlxR physiological function have been hindered by the severe growth defects of a glxR mutant. This study comprehensively identified the GlxR regulon by chromatin immunoprecipitation in conjunction with microarray (ChIP-chip) analyses. In total, 209 regions were detected as in vivo GlxR binding sites. Moreover, ChIP-chip analyses showed that GlxR was still able to interact with its target sites in a deletion mutant of cyaB, the sole adenylate cyclase gene in the genome, even though binding affinity was markedly decreased. To identify the direct GlxR targets, we immunoprecipitated DNA from a strain expressing a Strep-tag II-tagged GlxR-protein using an anti-Strep-tag II antibody. To investigate effect of depletion of cAMP by deletion of the cyaB gene, which encodes the sole adenylate cyclase in C. glutamicum, on GlxR binding in vivo, we immunoprecipitated DNA from a cyaB deletion strain expressing a Strep-tag II-tagged GlxR-protein using an anti-Strep-tag II antibody. Three or more independent biological replicates were generated in both cases.

2011-12-02 | E-GEOD-26870 | biostudies-arrayexpress

Chip-chip from C. glutamicum wild type expressing GlxR tagged with Strep Tag II and cyaB deletion strain expressing GlxR tagged with Strep Tag II.

2011-12-02 | GSE26870 | GEO

Tandem affinity purification of functional TAP-tagged proteins from human cells.

Project description:Tandem affinity purification (TAP) is a generic two-step affinity purification protocol for isolation of TAP-tagged proteins together with associated proteins. We used bacterial artificial chromosome to heterologously express TAP-tagged murine Sgo1 protein in human HeLa cells. This allowed us to test the functionality of the Sgo1-TAP protein by RNA interference-mediated depletion of the endogenous human Sgo1. Here, we present an optimized protocol for purification of TAP-tagged Sgo1 protein as well as KIAA1387 from HeLa cells with detailed instructions. The purification protocol can be completed in 1 day and it should be applicable to other proteins.

| S-EPMC2957861 | biostudies-literature

Transcription profiling by array of human MCF cells treated with estrogen

Project description:The estrogen receptor is the master transcriptional regulator of breast cancer phenotype and the archetype of a molecular therapeutic target. We mapped all estrogen receptor and RNA polymerase II binding sites on a genome-wide scale, identifying the authentic cis binding sites and target genes, in breast cancer cells. Combining this unique resource with gene expression data demonstrates distinct temporal mechanisms of estrogen-mediated gene regulation,particularly in the case of estrogen-suppressed genes. Furthermore, this resource has allowed the identification of cis-regulatory sites in previously unexplored regions of the genome and the cooperating transcription factors underlying estrogen signaling in breast cancer. Experiment Overall Design: This Series currently contains the gene expression data accompaning Carroll JS et al. Nature Genetics 38,1289-1297(2006). MCF7 cells were stimulated with 100 nM estrogen for 0, 3, 6, or 12h. All experiments were performed in triplicate.

| S-ECPF-GEOD-11324 | biostudies-other

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