Estrogen Receptor Transcriptional Regulation

MCF-7 cells were seeded onto 24-well plates at a density of 2 × 10⁵ cells/well and cotransfected with 1 μg of the 3× ERE TATA Luc (oestrogen-responsive element-containing luciferase reporter) construct (Addgene, http://www.addgene.org, plasmid 11354) [27 (link)] and 300 ng of the control pRL-TK plasmid (Promega Italia S.r.l., Milano, Italy) for normalization of transfection efficiency. The putative promoter region of the human FGFR2 gene (−1103 to +459 relative to the transcriptional initiation site) was amplified and inserted into a luciferase reporter vector, as previously described [26 (link)]. Transfections with ERE-luc or pKGFR construct were carried out in triplicate using Lipofectamine 2000 (Invitrogen) following manufacturer's instructions. Luciferase activities were determined with Dual Luciferase Reporter Assay System (Promega) 24 hrs after treatment, according to manufacturer's protocol.

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Ceccarelli S., D'Amici S., Vescarelli E., Coluccio P., Matricardi P., di Gioia C., Benedetti Panici P., Romano F., Frati L., Angeloni A, & Marchese C. (2014). Topical KGF treatment as a therapeutic strategy for vaginal atrophy in a model of ovariectomized mice. Journal of Cellular and Molecular Medicine, 18(9), 1895-1907.

Publication 2014

3× ERE TATA Luc pRL-TK plasmid Lipofectamine 2000 Dual Luciferase Reporter Assay System

After treatment Assay Fgfr2 Gene Human Lipofectamine 2000 Luciferase Mcf 7 cells Oestrogen Plasmid Promega Transcriptional initiation site Transfections Vector

Corresponding Organization : Sapienza University of Rome

Other organizations : Istituto Superiore di Sanità

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Variable analysis

independent variables

Transfection with 3× ERE TATA Luc construct
Transfection with pRL-TK plasmid
Transfection with putative promoter region of human FGFR2 gene (pKGFR construct)

dependent variables

Luciferase activities

control variables

MCF-7 cell density (2 × 10^5 cells/well)
Transfection method (Lipofectamine 2000)

controls

Positive control: 3× ERE TATA Luc (oestrogen-responsive element-containing luciferase reporter) construct
Negative control: Not explicitly mentioned

Annotations

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