PDF(Pubmed)
Abstract：
Macroautophagy/autophagy is a regulated cellular degradation process essential as a pro-survival mechanism and integral to the regulation of diverse cellular processes in eukaryotes. During cellular stress and nutrient sensing, SQSTM1/p62 (sequestosome 1) functions as a key receptor for selective autophagy by shuttling ubiquitinated cargoes toward autophagic degradation making it a useful marker for monitoring autophagic flux. We present a straightforward and rapid flow cytometric assay for the quantitative measurement of intracellular SQSTM1 with improved sensitivity to conventional immunoblotting and with the benefit of higher throughput and reduced requirements for starting cellular materials for adequate analysis. We demonstrate that flow cytometry is able to detect similar trends in the measurement of intracellular SQSTM1 levels following serum starvation, genetic manipulations, and bafilomycin A1/chloroquine treatments. The assays utilizes readily available reagents and equipment without the need for transfection and utilizes standard flow cytometry equipment. In the present studies, expression of reporter proteins was applied to a range of SQSTM1 expression levels generated by genetic and chemical manipulation in both mouse as well as human cells. In combination with appropriate controls and attention to cautionary issues, this assay offers the ability to assess an important measure of autophagic capacity and flux.Abbreviations: ATG5: autophagy related 5 ATG7: autophagy related 7 BafA: bafilomycin A1 BMDM: bone marrow-derived macrophages CQ: chloroquine EBV: Epstein-Barr Virus EDTA: ethylenediaminetetraacetic acid FBS: fetal bovine serum gMFI: geometric mean fluorescent intensity HD: healthy donor MAP1LC3/LC3/Atg8: microtubule associated protein 1 light chain 3 MedianFI: median fluorescent intensity NTC: non-target control PBMC: peripheral blood mononuclear cells RPMI: Roswell Park Memorial Institution SQSTM1/p62: sequestosome 1 WT: wild type.
摘要：
巨自噬/自噬是一种受调节的细胞降解过程，是一种促生存机制，是调节真核生物多种细胞过程的不可或缺的组成部分。在细胞应激和营养感知期间，SQSTM1/p62（隔离体1）通过将泛素化的货物穿梭于自噬降解而充当选择性自噬的关键受体，使其成为监测自噬通量的有用标记。我们提出了一种简单而快速的流式细胞术，用于定量测量细胞内SQSTM1，具有对常规免疫印迹的改进敏感性，并具有更高的通量和减少对起始细胞材料进行充分分析的要求的益处。我们证明，流式细胞术能够检测血清饥饿后细胞内SQSTM1水平测量的相似趋势，遗传操作,和巴弗洛霉素A1/氯喹治疗。该测定利用容易获得的试剂和设备而无需转染,并利用标准流式细胞术设备。在目前的研究中,将报告蛋白的表达应用于小鼠和人类细胞中通过遗传和化学操作产生的一系列SQSTM1表达水平。结合适当的控制和对警示问题的关注，该试验提供了评估自噬能力和通量的重要指标的能力。缩写：ATG5：自噬相关5ATG7：自噬相关7BafA：bafilomycinA1BMDM：骨髓来源的巨噬细胞CQ：氯喹EBV：爱泼斯坦-巴尔病毒EDTA：乙二胺四乙酸FBS：牛胎儿血清gMFI：平均几何荧光强度：健康供体MAP1LHD/LC3/Attughn中心基因基因对照8：MENALC
