Lin Hui-Hsien, Robertson Kelly L, Lellupitiyage Don Sujeewa S, Taylor Stephanie R, Farkas Michelle E
Department of Chemistry, University of Massachusetts Amherst, Amherst, MA, United States.
Department of Biochemistry and Molecular Biology, University of Massachusetts Amherst, Amherst, MA, United States.
Methods Enzymol. 2020;639:115-140. doi: 10.1016/bs.mie.2020.04.011. Epub 2020 Apr 30.
Circadian rhythms are critical regulators of many physiological and behavioral functions. The use and abilities of small molecules to affect oscillations have recently received significant attention. These manipulations can be reversible and tunable, and have been used to study various biological mechanisms and molecular properties. Here, we outline procedures for assessment of cellular circadian changes following treatment with small molecules, using luminescent reporters. We describe reporter generation, luminometry experiments, and data analysis. Protocols for studies of accompanying effects on cells, including motility, viability, and anchorage-independent proliferation assays are also presented. As examples, we use indirubin-3'-oxime and two derivatives, 5-iodo-indirubin-3'-oxime and 5-sulfonic acid-indirubin-3'-oxime. In this case study, we analyze effects of these compounds on Bmal1 and Per2 (positive and negative core circadian elements) oscillations and provide step-by-step protocols for data analysis, including removal of trends from raw data, period estimations, and statistical analysis. The reader is provided with detailed protocols, and guidance regarding selection of and alternative approaches.
昼夜节律是许多生理和行为功能的关键调节因子。小分子影响振荡的用途和能力最近受到了广泛关注。这些操作可以是可逆的和可调的，并已被用于研究各种生物学机制和分子特性。在这里，我们概述了使用发光报告基因评估小分子处理后细胞昼夜变化的程序。我们描述了报告基因的生成、发光测定实验和数据分析。还介绍了研究对细胞伴随影响的方案，包括运动性、活力和非锚定依赖性增殖测定。作为示例，我们使用靛玉红 - 3'-肟及其两种衍生物，5-碘靛玉红 - 3'-肟和5-磺酸靛玉红 - 3'-肟。在这个案例研究中，我们分析了这些化合物对Bmal1和Per2（昼夜节律核心正负元件）振荡的影响，并提供了数据分析的分步方案，包括从原始数据中去除趋势、周期估计和统计分析。为读者提供了详细的方案以及关于选择和替代方法的指导。
