FKHR Redistribution Assay

• Designed to assay compounds for their ability to modulate nuclear translocation of FKHR (FOXO1)
• 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 FKHR is regulated via its phosphorylation by the protein kinase Akt that is part of the phosphoinositide 3-kinase (PI3K) signaling pathway. Phosphorylated FKHR is sequestered in its inactive form within the cytosol by the so-called 14-3-3 protein. Un-phosphorylated and active FKHR reside 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 FKHR phosphorylation and cytoplasmic sequestering, eventually resulting in nuclear accumulation of FKHR.

Figure 1. Translocation of FKHR-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 concentration response curve in the FKHR Redistribution assay. Concentration response was measured in 9 point half log dilution series of wortmannin. Cells were incubated with wortmannin for 60 min. Cells were then fixed and the nucleus to cytoplasm translocation was measured using the Cellomics ArrayScan V^TI Reader and the RedistributionV3 BioApplication. % activity was calculated relative to the positive (150 nM wortmannin) and negative control (0.25% DMSO). The EC₅₀ of wortmannin is approximately 9 nM.

Figure 3. Concentration response curves in the FKHR Redistribution assay. Concentration response was measured in 9 point 2x dilution series of Leptomycin B (nuclear export inhibitor), LY294009 (PI3K inhibitor)and an Akt inhibitor. Cells were incubated with test 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 (150 nM wortmannin) and negative control (0.25% DMSO). The EC₅₀ values are: Leptomycin EC₅₀= 2.7 nM, LY294002 EC₅₀=4.7 μM and Akt inhibitor EC₅₀ = 22.

Figure 4. Western blot showing regulation of FKHR Redistribution cells. FKHR Redistribution cells were incubated with DMSO, wortmannin or an Akt inhibitor [4] for 60 min. Cells were then lysed and cell extracts were run on western blot. Akt polyclonal and FKHR-EGFP indicate the total amount of Akt1-3 and FKHR-EGFP in the assay cell line, respectively. P-S473-Akt indicates the amount of Akt that is activated by S473 phosphorylation. This activation is fully inhibited by wortmannin and the Akt inhibtor. P-S256-FKHR-EGFP indicates the amount of FKHR-EGFP that is phosphorylated on S256 (Akt site). FKHR phosphorylation is fully inhibited by wortmannin and partly inhibited by the Akt inhibitor.