Multiplexed Dual-Color Fluorescence-Based Distinction Between Nuclear Trapping and Translocation of FOXO3

FOXO3 is a transcription factor that mainly exerts its functions in the cell nucleus. The amino acid sequence of FOXO3 contains a nuclear localization sequence (NLS) and a nuclear export sequence (NES) allowing for nuclear/cytoplasmic shuttling that plays an important role in regulating FOXO3 activity. Nuclear accumulation of FOXO3 proteins can be the result of translocation to the nucleus triggered by upstream regulatory input or trapping of FOXO3 within the nucleus through the inhibition of its nuclear export via the receptor CRM1. In order to distinguish these two modes of FOXO3 activation, we have generated a multiplexed assay. The development of this platform includes a reporter cell line that monitors CRM1 activity by using RFP-labeled HIV-1 Rev. protein with a strong heterologous NES. Simultaneously, the intracellular localization of FOXO3 can be monitored by a second cell line stably expressing GFP-FOXO3. Here we describe a detailed protocol on how to co-culture these reporter cell lines and use them to interrogate compound-induced FOXO3 activation in order to understand the mode of action.