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Abstract：
Signaling pathways are involved in key cellular functions from embryonic development to pathological conditions, with a pivotal role in tissue homeostasis and transformation. Although most signaling pathways have been intensively examined, most studies have been carried out in murine models or simple cell culture. We describe the dissection of the TGF-β signaling pathway in human tissue using CRISPR-Cas9 genetically engineered human keratinocytes (N/TERT-1) in a 3D organotypic skin model combined with quantitative proteomics and phosphoproteomics mass spectrometry. The use of human 3D organotypic cultures and genetic engineering combined with quantitative proteomics and phosphoproteomics is a powerful tool providing insight into signaling pathways in a human setting. The methods are applicable to other gene targets and 3D cell and tissue models. Key features • 3D organotypic models with genetically engineered human cells. • In-depth quantitative proteomics and phosphoproteomics in 2D cell culture. • Careful handling of cell cultures is critical for the successful formation of the organotypic cultures. • For complete details on the use of this protocol, please refer to Ye et al. 2022.
摘要：
信号通路参与从胚胎发育到病理状态的关键细胞功能，在组织稳态和转化中起关键作用。尽管大多数信号通路都经过了深入的研究，大多数研究是在小鼠模型或简单的细胞培养中进行的。我们描述了使用CRISPR-Cas9基因工程人类角质形成细胞（N/TERT-1）在3D器官型皮肤模型中结合定量蛋白质组学和磷酸蛋白质组学质谱的人组织中TGF-β信号通路的解剖。使用人类3D器官型培养和基因工程结合定量蛋白质组学和磷酸蛋白质组学是一种强大的工具，可以深入了解人类环境中的信号通路。该方法适用于其他基因靶标以及3D细胞和组织模型。关键特征•具有基因工程人类细胞的3D器官型模型。•2D细胞培养中的深度定量蛋白质组学和磷酸蛋白质组学。•细胞培养物的小心处理对于器官型培养物的成功形成是关键的。•Forcompletedetailsontheuseofthisprotocol,请参阅Ye等人。2022年。
