FOXO4/AFX Redistribution Assay

• Designed to assay compounds for their ability to modulate nuclear translocation of Foxo4/AFX
• 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

FOXO4/AFX was first described as acute-lymphocytic-leukaemia-1 fused gene from chromosome X and is part of the Forkhead transcription factor family, which consists of FOXO1, FOXO3a, and FOXO4 (FKHR, FKHRL1, and AFX, respectively). Forkhead transcription factors function as key regulators of insulin signaling, cell cycle progression and apoptosis downstream of phosphoinositide 3-kinase (PI3K). They act as classical transcriptional activators and share DNA-binding specificity to a core consensus site called the Forkhead-responsive element. Target genes include pro-apoptotic proteins (e.g. Fas Ligand and Bim) and cell cycle inhibitors (e.g. p27, p130 and GADD45). In growing cells, Forkheads are kept inactive through Akt-mediated phosphorylation of three conserved serines and threonines. Phosphorylation of these residues causes binding to 14-3-3 proteins in the nucleus followed by nuclear export and cytoplasmic retention. Nuclear export of Forkhead proteins is dependent on the classical NES/Crm1 pathway. Inactive FOXO4/AFX is cytoplasmic but is rapidly imported to the nucleus upon inactivation of the PI3K/Akt pathway. Foxo4/AFX nuclear translocation can be promoted by the PI3K inhibitor wortmannin.

Figure 1. Translocation of FOXO4-EGFP. Cells were treated with 150 nM wortmannin for 1 hr (right panel) or untreated (DMSO control, left panel). Arrows indicate the nuclear localization detected by the image analysis algorithm.

Figure 2. Wortmannin concentration response in the FOXO4/AFX Redistribution assay. Concentration response was measured in 9 point half log dilution series (n=16). Cells were incubated with wortmannin for 1 hr. Cells were then fixed and the nucleus to cytoplasm translocation was measured using the Cellomics ArrayScan V^TI Reader and the Redistribution V3 BioApplication. % activity was calculated relative to the positive (150 nM wortmannin) and negative control (0.25% DMSO). The EC₅₀ of wortmannin is ~7nM.