Sols-Morreale Biomedical Research Institute (IIBM), Spanish National Research Council (CSIC), Universidad Autónoma de Madrid (UAM), Madrid, Spain.
ABC-RI, Algarve Biomedical Center Research Institute, Algarve Biomedical Center, Faro, Portugal.
Methods Mol Biol. 2025;2871:163-170. doi: 10.1007/978-1-0716-4217-7_15.
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.
FOXO3 是一种转录因子，主要在细胞核中发挥作用。FOXO3 的氨基酸序列包含一个核定位序列（NLS）和一个核输出序列（NES），允许核/细胞质穿梭，这在调节 FOXO3 活性中起着重要作用。FOXO3 蛋白的核积累可能是由于上游调节输入触发的向核易位，或者通过抑制其核输出通过受体 CRM1 将 FOXO3 困在核内而导致的。为了区分 FOXO3 激活的这两种模式，我们生成了一个多重分析。该平台的开发包括一个报告细胞系，该细胞系通过使用带有强异源 NES 的 RFP 标记的 HIV-1 Rev. 蛋白来监测 CRM1 活性。同时，可以通过稳定表达 GFP-FOXO3 的第二细胞系来监测 FOXO3 的细胞内定位。在这里，我们描述了一个详细的方案，说明如何共培养这些报告细胞系，并使用它们来研究化合物诱导的 FOXO3 激活，以了解其作用模式。
