Ribosome biogenesis is initiated in the nucleolus, a cell condensate essential to gene expression, whose morphology informs cancer pathologists on the health status of a cell. Here, we describe a protocol for assessing, both qualitatively and quantitatively, the involvement of trans-acting factors in the nucleolar structure. The protocol involves use of siRNAs to deplete cells of factors of interest, fluorescence imaging of nucleoli in an automated high-throughput platform, and use of dedicated software to determine an index of nucleolar disruption, the iNo score. This scoring system is unique in that it integrates the five most discriminant shape and textural features of the nucleolus into a parametric equation. Determining the iNo score enables both qualitative and quantitative factor classification with prediction of function (functional clustering), which to our knowledge is not achieved by competing approaches, as well as stratification of their effect (severity of defects) on nucleolar structure. The iNo score has the potential to be useful in basic cell biology (nucleolar structure-function relationships, mitosis, and senescence), developmental and/or organismal biology (aging), and clinical practice (cancer, viral infection, and reproduction). The entire protocol can be completed within 1 week.

中文翻译：

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使用iNo分数将正常人与改变的核仁形态区分开来，并应用于基础细胞生物学以及在人类疾病诊断中的潜力。

核糖体的生物发生在核仁中启动，核仁是基因表达所必需的细胞凝缩物，其形态告诉癌症病理学家有关细胞健康状况的信息。在这里，我们描述了一种用于定性和定量评估反式作用因子参与核仁结构的协议。该协议涉及使用siRNA耗尽感兴趣因子的细胞，在自动化的高通量平台上对核仁进行荧光成像，并使用专用软件确定核仁破坏指数iNo评分。该评分系统的独特之处在于，它将核仁的五个最有区别的形状和纹理特征整合到一个参数方程式中。确定iNo分数可实现对功能预测（功能聚类）的定性和定量因素分类，据我们所知，这是无法通过竞争方法实现的，而且无法对其作用（对缺陷的严重程度）进行分层。iNo评分可能在基础细胞生物学（核仁结构-功能关系，有丝分裂和衰老），发育和/或生物生物学（衰老）以及临床实践（癌症，病毒感染和生殖）中有用。整个协议可以在1周内完成。iNo评分可能在基础细胞生物学（核仁结构-功能关系，有丝分裂和衰老），发育和/或生物生物学（衰老）以及临床实践（癌症，病毒感染和生殖）中有用。整个协议可以在1周内完成。iNo评分可能在基础细胞生物学（核仁结构-功能关系，有丝分裂和衰老），发育和/或生物生物学（衰老）以及临床实践（癌症，病毒感染和生殖）中有用。整个协议可以在1周内完成。