Abstract
BackgroundIndependent luciferase reporter assays and fluorescent translocation assays have been successfully used in drug discovery for several molecular targets. We developed U2transLUC, an assay system in which luciferase and fluorescent read-outs can be multiplexed to provide a powerful cell-based high content screening method.ResultsThe U2transLUC system is based on a stable cell line expressing a GFP-tagged FOXO transcription factor and a luciferase reporter gene under the control of human FOXO-responsive enhancers. The U2transLUC assay measures nuclear-cytoplasmic FOXO shuttling and FOXO-driven transcription, providing a means to analyze these two key features of FOXO regulation in the same experiment. We challenged the U2transLUC system with chemical probes with known biological activities and we were able to identify compounds with translocation and/or transactivation capacity.ConclusionCombining different biological read-outs in a single cell line offers significant advantages over conventional cell-based assays. The U2transLUC assay facilitates the maintenance and monitoring of homogeneous FOXO transcription factor expression and allows the reporter gene activity measured to be normalized with respect to cell viability. U2transLUC is suitable for high throughput screening and can identify small molecules that interfere with FOXO signaling at different levels.
Highlights
Independent luciferase reporter assays and fluorescent translocation assays have been successfully used in drug discovery for several molecular targets
The U2transLUC assay facilitates the maintenance and monitoring of homogeneous Forkhead box O (FOXO) transcription factor expression and allows the reporter gene activity measured to be normalized with respect to cell viability
U2transLUC is suitable for high throughput screening and can identify small molecules that interfere with FOXO signaling at different levels
Summary
Independent luciferase reporter assays and fluorescent translocation assays have been successfully used in drug discovery for several molecular targets. FOXO transcription factors have been proposed to act as bona fide tumor suppressors due to their inhibitory effects on cell cycle and survival [4], properties mediated by their binding as monomers to consensus DNA binding sites. Their transcriptional activity is governed by a network of signaling events, the best recognized of which is the phosphorylation of FOXO proteins at three highly conserved serine and threonine residues by Akt that provokes its association with 14-3-3 protein and in turn, the nuclear exclu-. Systematic chemical genetic or loss of function studies to investigate the complex regulation of FOXO factors have been limited only to certain aspects [9]
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