Human retinal pigment epithelial-1 (RPE-1) cells are increasingly being used as a model to study mitosis because they represent a non-transformed alternative to cancer cell lines, such as HeLa cervical adenocarcinoma cells. However, the lack of an efficient method to synchronize RPE-1 cells in mitosis precludes their application for large-scale biochemical and proteomics assays. Here, we report a protocol to synchronize RPE-1 cells based on sequential treatments with the Cdk4 and Cdk6 inhibitor PD 0332991 (palbociclib) and the microtubule-depolymerizing drug nocodazole. With this method, the vast majority (80-90%) of RPE-1 cells arrested at prometaphase and exited mitosis synchronously after release from nocodazole. Moreover, the cells fully recovered and re-entered the cell cycle after the palbociclib-nocodazole block. Finally, we show that this protocol could be successfully employed for the characterization of the protein-protein interaction network of the kinetochore protein Ndc80 by immunoprecipitation coupled with mass spectrometry. This synchronization method significantly expands the versatility and applicability of RPE-1 cells to the study of cell division and might be applied to other cell lines that do not respond to treatments with DNA synthesis inhibitors.

中文翻译：

---

人视网膜色素上皮-1 细胞有丝分裂的同步。

人视网膜色素上皮-1 (RPE-1) 细胞越来越多地被用作研究有丝分裂的模型，因为它们代表了癌细胞系（例如 HeLa 宫颈腺癌细胞）的非转化替代品。然而，缺乏同步 RPE-1 细胞有丝分裂的有效方法阻碍了它们在大规模生化和蛋白质组学检测中的应用。在这里，我们报告了一种基于 Cdk4 和 Cdk6 抑制剂 PD 0332991 (palbociclib) 和微管解聚药物诺考达唑序贯治疗来同步 RPE-1 细胞的方案。通过这种方法，绝大多数（80-90%）的RPE-1细胞在前期停滞，并在诺考达唑释放后同步退出有丝分裂。此外，在哌柏西利-诺考达唑阻断后，细胞完全恢复并重新进入细胞周期。最后，我们表明该方案可以通过免疫沉淀结合质谱成功地用于表征着丝粒蛋白 Ndc80 的蛋白质-蛋白质相互作用网络。这种同步方法显着扩展了 RPE-1 细胞在细胞分裂研究中的多功能性和适用性，并可能应用于对 DNA 合成抑制剂治疗没有反应的其他细胞系。