FKHRL1 Redistribution Assay

• Designed to assay compounds for their ability to modulate nuclear translocation of FKHRL1 (FOXO3)
• 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

Forkhead proteins comprise a highly conserved family of transcription factors named after the original forkhead gene in Drosophila. Forkhead transcription factor family members (FKHR, FKHRL1 and AFX) are known to control the expression of genes encoding proteins essential for insulin, apoptosis (e.g. Fas Ligand and Bim) and cell cycle (e.g. p27, p130 and GADD45) signaling. The activity of FKHRL1 is regulated via its phosphorylation by the protein kinase Akt that is part of the phosphoinositide 3-kinase (PI3K) signaling pathway. Phosphorylated FKHRL1 is sequestered in its inactive form within the cytosol by the so-called 14-3-3 protein. Unphosphorylated and active FKHRL1 resides in the nucleus. Furthermore, cellular localization of forkhead proteins is also dependent on the classical nuclear export sequence (NES)/Crm1 pathway. Wortmannin inhibits PI3K signalling and hereby hinders FKHRL1 phosphorylation and cytoplasmic sequestering, eventually resulting in nuclear accumulation of FKHRL1. In this assay wortmannin is used as reference compound. Test compounds are assayed for their ability to induce nuclear accumulation of FKHRL1.

Figure 1. Translocation of FKHRL1-EGFP in response to wortmannin. Cells were treated with 0.25% DMSO (left panel) or 300 nM wortmannin (right panel). Arrows indicate cytoplasm to nucleus translocation detected by the image analysis algorithm.

Figure 2. Wortmannin and LY294002 concentration response curves in the FKHRL1 Redistribution assay. Concentration response was measured in 9 point half log dilution series of wortmannin and LY294002. Cells were incubated with compound for 60 min. Cells were then fixed and the nucleus to cytoplasm translocation was measured using image analysis. % activity was calculated relative to the positive (300 nM wortmannin) and negative control (0.25% DMSO). The EC₅₀ of wortmannin is approximately 7.5 nM and the EC₅₀ of LY294002 is approximately 3.6 μM.