Abstract：
The epithelial-mesenchymal transition (EMT) plays an important role in development and cancer progression. Upon EMT, epithelial cells lose stable cell-cell adhesions and reorganize their cytoskeleton to acquire migratory activity. Recent data demonstrated that EMT drives cancer cells from the epithelial state to a hybrid epithelial/mesenchymal phenotype with retention of some epithelial markers (in particular, E-cadherin), which is important for cancer cell dissemination. In vitro studies of the effect of growth factors (in particular, epidermal growth factor (EGF)) on cultured cells can be highly advantageous for understanding the details of the early stages of EMT. The methods described in this chapter are intended for studying intermediate phenotypes of EMT. Time-lapse DIC microscopy is used for visualization of changes in morphology and motility of the cells stimulated with EGF. The transwell migration assay allows the evaluation of the migratory activity of the cells. Studying of dynamics of a fluorescently labeled actin-binding protein F-tractin-tdTomato using confocal microscopy allows detection of EGF-induced changes in the organization of the actin cytoskeleton. Live-cell imaging of cells stably expressing GFP-E-cadherin visualizes reorganization of stable tangential E-cadherin-based adherens junctions (AJs) into unstable radial AJs during the early stages of EMT.
摘要：
上皮-间质转化（EMT）在癌症的发展和进展中起着重要作用。在EMT时，上皮细胞失去稳定的细胞-细胞粘附并重组其细胞骨架以获得迁移活性。最近的数据表明，EMT驱动癌细胞从上皮状态到混合上皮/间质表型，保留一些上皮标志物（特别是，E-cadherin)，这对癌细胞传播很重要。生长因子作用的体外研究（特别是，培养细胞上的表皮生长因子（EGF））对于了解EMT早期阶段的细节非常有利。本章描述的方法旨在研究EMT的中间表型。延时DIC显微镜用于观察用EGF刺激的细胞的形态和运动性的变化。transwell迁移测定允许评估细胞的迁移活性。使用共聚焦显微镜研究荧光标记的肌动蛋白结合蛋白F-tractin-tdTomato的动力学，可以检测EGF诱导的肌动蛋白细胞骨架组织变化。稳定表达GFP-E-钙黏着蛋白的细胞的活细胞成像可在EMT的早期阶段将稳定的切向基于E-钙黏着蛋白的粘附连接（AJ）重组为不稳定的径向AJ。
