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

Carlos Amenabar1, Lucia Jimenez1, Cristiana Mourato2,3

  • 1Sols-Morreale Biomedical Research Institute (IIBM), Spanish National Research Council (CSIC), Universidad Autónoma de Madrid (UAM), Madrid, Spain.

Methods in Molecular Biology (Clifton, N.J.)
|November 20, 2024
PubMed
Summary

We developed a novel assay to distinguish between two activation pathways for the transcription factor FOXO3 (Forkhead box protein O3). This method uses reporter cell lines to monitor CRM1 activity and FOXO3 localization simultaneously.

Keywords:
Anticancer drugsAntiviral drugsBiosensorsCRM1Nuclear exportOrganoselenium compoundsSelinexor

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Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • FOXO3 (Forkhead box protein O3) is a transcription factor regulating cellular processes via nuclear-cytoplasmic shuttling.
  • FOXO3 activity is modulated by its nuclear localization sequence (NLS) and nuclear export sequence (NES).
  • Nuclear accumulation of FOXO3 can occur through nuclear translocation or inhibition of nuclear export via CRM1.

Purpose of the Study:

  • To develop a multiplexed assay to differentiate between FOXO3 activation mechanisms.
  • To enable the study of compound-induced FOXO3 activation and its underlying mode of action.

Main Methods:

  • Generation of a reporter cell line expressing RFP-labeled HIV-1 Rev. protein to monitor CRM1 activity.
  • Development of a second cell line stably expressing GFP-FOXO3 to track intracellular localization.
  • Co-culture of both reporter cell lines for simultaneous monitoring.

Main Results:

  • The developed platform allows for the simultaneous assessment of CRM1 activity and FOXO3 localization.
  • This multiplexed assay can be used to interrogate compound effects on FOXO3 activation pathways.

Conclusions:

  • The described protocol provides a robust method for distinguishing FOXO3 activation modes.
  • This tool facilitates a deeper understanding of FOXO3 regulation and drug discovery efforts targeting this pathway.