Effects of TCDCA-FXR on obesity-induced endothelial dysfunction and hypertension III

To investigate the role of TCDCA-FXR in endothelial cells, we performed CUT&TAG in HUVECs. Overall design: The transcription factor ATF4 has been reported to regulate the expression of the key enzymes involved in serine metabolism (PHGDH, PSAT1, and SHMT2) and one-carbon metabolism (MTHFD2, ALDH1L2). Knockdown of ATF4 in HUVECs treated with TCDCA led to a significant downregulation in the mRNA levels of PHGDH, PSAT1, and SHMT2. As a nuclear receptor and transcription factor, FXR can regulate gene expression upon TCDCA activation. To determine if FXR directly regulates ATF4 transcription, we performed cleavage under targets and tagmentation (CUT&TAG) assay using Flag antibody in HUVECs overexpressing Flag-tagged FXR. TCDCA treatment resulted in reduced nuclear enrichment of PHB1, while TUDCA maintained strong nuclear colocalization of FXR and PHB1. To further evaluated the role of PHB1, we performed CUT&TAG assay to evaluate the DNA-binding profiles of PHB1 in ECs

为探究牛磺鹅去氧胆酸-法尼醇X受体（TCDCA-FXR）在内皮细胞中的作用，我们在人脐静脉内皮细胞（HUVECs）中开展了靶标切割与标签化（CUT&TAG）实验。实验整体设计如下：已有研究表明，转录因子ATF4可调控丝氨酸代谢关键酶（PHGDH、PSAT1及SHMT2）与一碳代谢关键酶（MTHFD2、ALDH1L2）的表达。在经TCDCA处理的HUVECs中敲低ATF4，会显著下调PHGDH、PSAT1与SHMT2的mRNA水平。作为核受体兼转录因子的法尼醇X受体（FXR），可在TCDCA激活后调控基因表达。为验证FXR是否可直接调控ATF4的转录，我们在过表达Flag标签FXR的HUVECs中，使用Flag抗体开展了靶标切割与标签化（CUT&TAG）实验。TCDCA处理会降低PHB1的核富集水平，而牛磺熊去氧胆酸（TUDCA）可维持FXR与PHB1的强核共定位。为进一步评估PHB1的作用，我们开展了靶标切割与标签化（CUT&TAG）实验，以分析内皮细胞中PHB1的DNA结合谱。