Rapid and synchronous chemical induction of replicative-like senescence via a small molecule inhibitor [bulk RNA-seq]

Cellular senescence is now acknowledged as a key contributor to organismal ageing and late-life disease. Although popular, the study of senescence in vitro can be complicated by the prolonged and asynchronous timing of cells committing to it and its paracrine effects. To address these issues, we repurposed the small molecule inhibitor inflachromene (ICM) to induce senescence to human primary cells. Within six days of treatment with ICM, senescence hallmarks, including the nuclear eviction of HMGB1 and -B2, are uniformly induced across the cell population. By comparing various high throughput datasets from ICM-induced and replicative senescence, we uncovered significant similarities between the two states. Notably, ICM suppresses the proinflammatory secretome associated with senescence, thus alleviating most paracrine effects. In summary, ICM rapidly and synchronously induces a senescence-like phenotype that allows us to study its core regulatory program without any confounding heterogeneity. Overall design: Micro-C is applied to 4 low passage IMR90 wild type samples and 4 low passage ICM-treated samples. RNA-seq and Ribo-Seq are applied to 4 low passage IMR90 wild type samples, 4 high passage IMR90 senescent samples and to 2 low passage ICM-treated samples. scRNA-seq is applied to 1 low passage IMR90 wild type samples, 1 high passage IMR90 senescent sample and to 1 low passage ICM-treated sample. FactoryRNA-seq is applied to 2 low passage IMR90 wild type samples, 2 low passage 3 days ICM-treated samples, 2 low passage 6 days ICM-treated samples and 2 low passage metaICM-treated samples. Cut&tag for CTCF and SMC1 are applied to 1 low passage IMR90 wild type sample, 1 high passage IMR90 senescent sample and to 1 low passage ICM-treated sample.

细胞衰老现已被证实为机体衰老与晚年疾病的关键致病因素之一。尽管体外衰老研究应用广泛，但细胞进入衰老程序的时间冗长且异步，加之其旁分泌效应，常导致研究复杂化。为解决上述问题，我们对小分子抑制剂inflachromene（ICM）进行了重新开发利用，用以诱导人类原代细胞发生衰老。经ICM处理后的6天内，细胞群体可同步诱导出各类衰老标志物，包括HMGB1与HMGB2的核迁出现象。通过对比ICM诱导衰老与复制性衰老（replicative senescence）的多组高通量数据集，我们发现二者具有显著的特征相似性。值得注意的是，ICM可抑制衰老相关的促炎分泌组，从而有效减轻绝大多数旁分泌效应带来的干扰。综上，ICM可快速且同步地诱导出类衰老表型，使我们能够在不受混杂异质性干扰的前提下，研究衰老的核心调控程序。

整体实验设计：
1. 对4株低传代IMR90野生型样本与4株低传代ICM处理样本开展Micro-C测序；
2. 对4株低传代IMR90野生型样本、4株高传代衰老IMR90样本及2株低传代ICM处理样本进行RNA测序（RNA-seq）与核糖体测序（Ribo-Seq）；
3. 对1株低传代IMR90野生型样本、1株高传代衰老IMR90样本及1株低传代ICM处理样本进行单细胞RNA测序（scRNA-seq）；
4. 对2株低传代IMR90野生型样本、2株低传代ICM处理3天样本、2株低传代ICM处理6天样本及2株低传代metaICM处理样本进行FactoryRNA-seq检测；
5. 对1株低传代IMR90野生型样本、1株高传代衰老IMR90样本及1株低传代ICM处理样本开展CTCF与SMC1的Cut&tag实验。