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High-Throughput Image-Based Screening to Identify Chemical Compounds Capable of Activating FOXO

  • Marta Barradas
  • Wolfgang Link
  • Diego Megias
  • Pablo J. Fernandez-Marcos
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1890)

Abstract

FOXO proteins are transcription factors with important roles in the regulation of the expression of genes involved in cell growth, proliferation, differentiation, and longevity. FOXO proteins are active in the nucleus but, upon post-translational modification they form a docking site for 14-3-3 proteins and are translocated to the cytoplasm where they are inactive.

We make use of this regulatory mechanism of FOXO proteins to develop an image-based high-throughput screening platform to detect compounds that regulate FOXO3 subcellular localization. This system has proven a powerful tool to isolate inhibitors of proteins upstream of FOXO, such as PI3K inhibitors.

Key words

Forkhead box O proteins–FOXO Nucleus-cytoplasm translocation High-content screening 

Notes

Acknowledgments

We thank Lola Martinez for her assistance in Flow Cytometry. Work in the laboratory of P.J.F.-M. was funded by the IMDEA Food, the Spanish Association Against Cancer (aecc) and the FBBVA and Ramon Areces Foundations. This work was supported by Fundação para a Ciência e a Tecnologia (FCT) Research Center Grant UID/BIM/04773/2013 Centre for Biomedical Research 1334. Work in the laboratory of WL was funded by a research grant from Liga Portuguesa Contra o Cancro – Núcleo Regional do Sul (LPCC/NRS), Terry Fox.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Marta Barradas
    • 1
  • Wolfgang Link
    • 2
  • Diego Megias
    • 3
  • Pablo J. Fernandez-Marcos
    • 1
  1. 1.Bioactive Products and Metabolic Syndrome GroupMadrid Institute of Advanced Studies–IMDEA Food, CEI UAM+CSICMadridSpain
  2. 2.Instituto de Investigaciones Biomédicas “Alberto Sols” (CSIC-UAM). Arturo Duperier 4. 28029-Madrid, SpainMadridSpain
  3. 3.Confocal Microscopy Unit, Biotechnology ProgramSpanish National Cancer Research Centre (CNIO)MadridSpain

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