Estrogen Receptor alpha (ERα) Redistribution Assay

• Designed to assay compounds for their ability to modulate accumulation of ERα in nuclear foci
• Coupled to EGFP for easy monitoring of the cellular translocation event
• Robust cell-based assay for use in high content analysis and fluorescence microscope applications

• Biologically relevant data: Compounds tested in a cellular environment
• Validated: Functionally tested cells provided with an optimized assay protocol
• Easy to use: Just plate cells, add compounds, and image

Estrogen is a regulator of normal endocrine functions. Signal transduction induced by estrogens such as estradiol, the main endogenous human estrogen, is mediated by the estrogen receptor (ER). ER is a nuclear receptor that upon ligand binding organizes into homo- and heterodimers of the ERα and ERβ subtypes. Following ligand binding, ER acts as a transcription factor and regulates expression of several target genes such as cyclin D1 and IGF-1. Estrogens and estrogen receptors are implicated in development and progression of breast cancer. Moreover, environmental chemical contaminants with estrogenic activity are suggested to promote reproductive disorders. The EC₅₀ of 17β-estradiol in the assay is approximately 0.3 nM and corresponds well with EC₅₀ values reported by others.

Figure 1. Nuclear foci formation of EGFP-ERα. Cells were untreated (DMSO control, left panel) or treated with 30 nM 17β-estradiol for 20 hr (right panel). Arrows indicate the nuclear foci containing EGFP-ERα that are detected by the image analysis algorithm.

Figure 2. Concentration response curves in the Estrogen Receptor alpha assay: A) 17β-estradiol concentration response in the ERα agonist assay (n=16). The EC₅₀ is approximately 0.3 nM. Concentration response was measured in 9 point half log dilution series. Cells were treated with 17β-estradiol for 20 hr. Cells were then fixed and nuclear foci formation was measured using the Cellomics ArrayScan V^TI Reader and the SpotDetectorV3 BioApplication. % activity was calculated relative to the positive (30 nM 17β-estradiol) and negative control (0.25% DMSO).

B) ICI 182,780 concentration response in the ERα antagonist assay (n=16). The EC₅₀ is approximately 9 nM. Cells were treated with 17β-estradiol in the presence of a half log dilution series of ICI 182,780 for 20 hr. Cells were then fixed and nuclear foci formation was measured using the IN Cell Analyzer 3000 (GE Healthcare). % activity was calculated relative to the positive (1 µM ICI 182,780) and negative control (0.25% DMSO).