BRD4-mediated epigenetic regulation of endoplasmic reticulum-mitochondria contact sites is governed by the mitochondrial complex III

This is a high content drug screening dataset composed of images and single-cell analysis results from a 384-well plate The SPLICS reporter is composed of a split GFP1-10 barrel localized to the mitochondrial outer membrane, a P2A self-cleavage signal for equimolar expression, and the remaining beta11 fragment targeted to the ER membrane. Upon ERMCS formation within the 8-10 nm distance, the split-GFP reporter forms intact GFP. Doxycycline-inducible SPLICS reporter stable cell lines were generated via a three-plasmid PiggyBac transposase system. Cells were co-transfected with the plasmids using Lipofectamine 2000 transfection reagent, then the next day selected with 2 mg/ml of G418 (Geneticin) for 7 days. With the SPLICS integrated cells, mitochondrial TagRFP is expressed in them. For mitochondrial TagRFP labeling in cells, we utilized a lentiviral backbone and introduced a mitochondria matrix TagRFP (mitoTagRFP) to control for gene expression and monitor mitochondrial abundance. After mitoTagRFP integration, 100 ng/ml of doxycycline were used to induce SPLICS reporter expression, and GFP/TagRFP double positive cells were sorted on Bigfoot Spectral Cell Sorter (Invitrogen) to isolate GFP/TagRFP positive clones. Clones were cultured and screened for accurate localization of SPLICS reporter to mitochondria and ER via live cell imaging and for normal mitochondrial oxygen consumption rate. Probe induction did not have a deleterious effect on cell number or increasing green fluorescence for at least 72 hours of reporter induction. For ERMCS analysis with SPLICS, cells will be induced with 100 ng/ml of doxycycline for minimally 24 hr before treatment or analysis.本数据集为基于384孔板的图像与单细胞分析结果构建的高内涵药物筛选数据集。 SPLICS报告基因（SPLICS reporter）由定位于线粒体外膜的拆分式GFP1-10桶状结构、用于实现等摩尔表达的P2A自剪切信号肽，以及靶向内质网膜的剩余β11片段组成。当内质网-线粒体接触位点（ERMCS）在8-10 nm的距离内形成时，该拆分GFP报告基因可组装为完整的GFP蛋白。 我们通过三质粒PiggyBac转座酶系统构建了多西环素诱导型SPLICS报告基因稳定细胞系。使用Lipofectamine 2000转染试剂将质粒共转染至细胞中，于转染次日加入2 mg/mL的G418（遗传霉素）筛选7天。整合有SPLICS报告基因的细胞可表达线粒体靶向TagRFP。为实现细胞内线粒体TagRFP标记，我们采用慢病毒载体骨架，引入线粒体基质靶向TagRFP（mitoTagRFP）以监控基因表达水平与线粒体丰度。 在mitoTagRFP整合完成后，使用100 ng/mL的多西环素诱导SPLICS报告基因表达，并通过Invitrogen公司的Bigfoot光谱细胞分选仪分选出GFP与TagRFP双阳性细胞克隆。对获得的克隆进行培养，并通过活细胞成像验证SPLICS报告基因在线粒体与内质网的精准定位，同时检测其线粒体氧消耗速率是否正常。实验结果表明，在报告基因诱导至少72小时内，探针诱导不会对细胞数量产生有害影响，也不会增加绿色荧光信号。 若需通过SPLICS进行ERMCS分析，需在处理或开展分析前，先用100 ng/mL的多西环素诱导细胞至少24小时。