Ma Richard Y M, Tong Tommy H K, Leung Wai Ying, Yao Kwok-Ming
Faculty of Medicine, Department of Biochemistry, The University of Hong Kong, Pokfulam, Hong Kong, China.
Methods Mol Biol. 2010;647:113-23. doi: 10.1007/978-1-60761-738-9_6.
The proliferation-associated transcription factor FOXM1 is essential for cell cycle progression into mitosis. Using synchronized human fibroblasts we detected, by immunostaining, that FOXM1 is localized predominantly in the cytoplasm in cells at late-G1 and S phases. Nuclear translocation occurs just before progression into the G2/M phase of the cell cycle and requires activity of the Raf/MEK/MAPK signaling pathway. Using pharmacological modulators, we demonstrated that activity of the Raf/MEK/MAPK pathway is both necessary and sufficient for the nuclear translocation of FOXM1. Consistent with FoxM1c being the major isoform expressed in proliferating fibroblasts, constitutively active MEK1 enhances the transactivating effect of FOXM1c, but not FOXM1b, on the cyclin B1 promoter in transient reporter assays. Here, we describe in detail the methods involved in generating these findings, which support the notion that FOXM1 is an effector of Raf/MEK/MAPK signaling in G2/M regulation.
增殖相关转录因子FOXM1对于细胞周期进入有丝分裂至关重要。我们使用同步化的人成纤维细胞，通过免疫染色检测到，在G1晚期和S期的细胞中，FOXM1主要定位于细胞质中。核转位恰好在细胞周期进入G2/M期之前发生，并且需要Raf/MEK/MAPK信号通路的活性。使用药理学调节剂，我们证明Raf/MEK/MAPK通路的活性对于FOXM1的核转位既必要又充分。与FoxM1c是增殖性成纤维细胞中表达的主要异构体一致，在瞬时报告基因测定中，组成型活性MEK1增强了FOXM1c而非FOXM1b对细胞周期蛋白B1启动子的反式激活作用。在这里，我们详细描述了产生这些发现所涉及的方法，这些发现支持了FOXM1是G2/M调节中Raf/MEK/MAPK信号的效应器这一观点。
