Equipe labellisée par la Ligue contre le cancer, Université de Paris, Sorbonne Université, INSERM UMR1138, Centre de Recherche des Cordeliers, Paris, France; Metabolomics and Cell Biology Platforms, Gustave Roussy, Villejuif, France; Université Paris Sud, Paris Saclay, Faculty of Medicine, Kremlin Bicêtre, Paris, France.
Equipe labellisée par la Ligue contre le cancer, Université de Paris, Sorbonne Université, INSERM UMR1138, Centre de Recherche des Cordeliers, Paris, France; Metabolomics and Cell Biology Platforms, Gustave Roussy, Villejuif, France.
Methods Cell Biol. 2022;172:67-82. doi: 10.1016/bs.mcb.2021.12.026. Epub 2022 Jan 17.
Anticancer drugs that suppress DNA-to-RNA transcription are particularly efficient in stimulating immunogenic cell death and hence eradicate malignant cells in a way that they will ignite an antitumor immune response. This is therapeutically relevant as it allows treatment response to last beyond drug discontinuation. For this reason, it is important to measure transcription inhibition in a precise fashion. Here, we detail two complementary assays for the assessment of transcription inhibition, one that detects the physical separation of fibrillarin and nucleolin by two-color immunofluorescence and another that measures the diminution of incorporated 5-ethynyl uridine (EU) into RNA, as revealed by click chemistry and the per-cell-intensity of a fluorescent signal.
抑制 DNA 到 RNA 转录的抗癌药物在刺激免疫原性细胞死亡方面特别有效，因此以一种能够引发抗肿瘤免疫反应的方式消灭恶性细胞。这在治疗上具有重要意义，因为它可以使药物停药后的治疗反应持续下去。出于这个原因，以精确的方式测量转录抑制是很重要的。在这里，我们详细介绍了两种互补的转录抑制评估检测方法，一种方法通过双色免疫荧光检测核仁蛋白和核仁小 RNA 结合蛋白（fibrillarin 和 nucleolin）的物理分离，另一种方法通过点击化学和荧光信号的每个细胞强度来测量掺入 RNA 中的 5-乙炔基尿嘧啶（EU）的减少。
