Image-based Identification of Chemical Compounds Capable of Trapping FOXO in the Cell Nucleus
Forkhead box O (FOXO) factors are tumor suppressor proteins commonly inactivated in human tumors. Furthermore, genetic variation within the FOXO3a gene is consistently associated with human longevity. FOXO proteins are usually inactivated by posttranslational modifications leading to cytoplasmic mislocalization. Therefore, the pharmacological activation by promoting nuclear localization of FOXOs is considered an attractive therapeutic approach to treat cancer and age-related diseases. We developed a cell-based imaging assay to screen for chemical agents capable of inhibiting the nuclear export and in turn trapping proteins that contain a nuclear export sequence including FOXO factors in the nucleus. The fluorescent signal of untreated assay cells localizes predominantly to the cytoplasm. Upon treatment with the nuclear export inhibitors the fluorescent-tagged reporter proteins appear as speckles in the nucleus. In a personalized medicine context, drugs capable of reactivating FOXO factors might be of enormous clinical value in human tumors in which these proteins are inactivated. Here, we describe the procedures for monitoring nuclear export which is suitable for high-throughput screening of compound collections.
Key wordsFOXO Nuclear export High-content screening CMR1
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. B. I. Ferreira is the recipient of a FCT 2014 research grant SFRH/BPD/100434/2014. S. Machado is the recipient of a ProRegem grant PD/BD/114258/2016.
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