Zanella Fabian, Rosado Aranzazú, Blanco Fernando, Henderson Beric R, Carnero Amancio, Link Wolfgang
Experimental Therapeutics Program, Centro Nacional de Investigaciones Oncologicas, Madrid, Spain.
Assay Drug Dev Technol. 2007 Jun;5(3):333-41. doi: 10.1089/adt.2007.058.
Intracellular localization is essential for the regulated activity of many signaling molecules associated with disease-relevant pathways. High content screening is a powerful technology to monitor the impact of small molecules or interfering RNAs on translocation of proteins within intact cells. Several assays have been developed to measure the nucleocytoplasmic shuttling of proteins like nuclear factor kappaB, FoxO, or nuclear factor of activated T-cells involved in distinct signaling networks. However, since all these proteins bear a leucine-rich nuclear export signal (NES), modulators of the NES-dependent export machinery can lead to misinterpretation of the assay readout. Here we report the generation of U2nesRELOC, a cell-based system for the identification of nuclear export inhibitors and specific silencers of the nuclear export machinery, and its adaptation to high throughput screening. The assay is based on mammalian cells stably expressing green fluorescent protein (GFP)-labeled Rev protein, which contains a strong heterologous NES. The fluorescent signal of untreated U2nesRELOC cells localizes exclusively to the cytoplasm. Upon treatment with the nuclear export inhibitor leptomycin B the GFP-labeled reporter protein accumulates rapidly in the cell nucleus. The assay has been adapted to 96-multiwell format and fully automated. Pilot experiments with a panel of 50 test compounds using three different concentrations per compound resulted in very consistent data sets with excellent reproducibility and an average Z' value of 0.76. In summary, U2nesRELOC is a cell-based nuclear export assay suitable for high throughput screening, providing counterscreens for pathway deconvolution.
细胞内定位对于许多与疾病相关途径相关的信号分子的调节活性至关重要。高内涵筛选是一种强大的技术，可用于监测小分子或干扰RNA对完整细胞内蛋白质转运的影响。已经开发了几种测定方法来测量参与不同信号网络的蛋白质（如核因子κB、FoxO或活化T细胞核因子）的核质穿梭。然而，由于所有这些蛋白质都带有富含亮氨酸的核输出信号（NES），NES依赖性输出机制的调节剂可能会导致对测定读数的错误解读。在这里，我们报告了U2nesRELOC的产生，这是一种基于细胞的系统，用于鉴定核输出抑制剂和核输出机制的特异性沉默剂，并将其应用于高通量筛选。该测定基于稳定表达绿色荧光蛋白（GFP）标记的Rev蛋白的哺乳动物细胞，该蛋白含有强大的异源NES。未处理的U2nesRELOC细胞的荧光信号仅定位于细胞质。在用核输出抑制剂雷帕霉素B处理后，GFP标记的报告蛋白迅速在细胞核中积累。该测定已适用于96孔板形式并完全自动化。使用一组50种测试化合物进行的先导实验，每种化合物使用三种不同浓度，得到了非常一致的数据集，具有出色的重现性，平均Z'值为0.76。总之，U2nesRELOC是一种适用于高通量筛选的基于细胞的核输出测定方法，为途径解卷积提供反筛选。
