Progesterone Receptor (PR) Redistribution Assay

• Designed to assay compounds for their ability to modulate nuclear translocation of the Progesterone Receptor
• 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

Progesterone is a female gonadal steroid hormone synthesized in the ovary. The progesterone receptor (PR) that specifically binds progesterone is member of the steroid receptor superfamily of nuclear receptors. The A form (PR-A) is a 164-amino acid N-terminally truncated version of the B form (PR-B). Unliganded PR is localized in the cytoplasm as well in the nucleus. Hormone binding to PR induces a conformational shift followed by dimerization into a complex that accumulates in the nucleus. In the nucleus PR activates and represses transcription of a range of target genes. Mice lacking PR exhibit reproductive abnormalities, incomplete mammary gland development,and impaired thymic function and sexual behavior. Clinically, progestational agents are used in oral contraceptives, menopausal hormone replacement, and treament of endometrial hyperplasia and breast cancer. The PR redistribution assay is based on human PR-B fused to EGFP and monitors translocation of EGFP-PR from the cytoplasm to the nucleus using progesterone as a reference compound. In addition to nuclear translocation, progesterone and other ligands induce accumulation of EGFP-PR in nuclear foci.

Figure 1. Nuclear translocation and foci formation of EGFP-PR. Cells were treated with 30 nM progesterone for 30 min (right panel) or untreated (DMSO control, left panel). This causes EGFP-PR to disappear from the cytoplasm and accumulate in the nucleus; this altered distribution is detected by the image analysis algorithm.

Figure 2. Concentration response curve in the PR assay: Progesterone concentration response in the PR agonist assay (n=16). The EC₅₀ is approximately 0.8 nM. Concentration response was measured in 9 point half log dilution series. Cells were treated with progesterone for 30 min. Cells were then fixed and nuclear foci formation was measured using the Cellomics ArrayScan V^TI Reader and the Redistribution V3 BioApplication. % activity was calculated relative to the positive (30 nM progesterone) and negative control (0.25% DMSO).