Monitoring autophagic flux by an improved tandem fluorescent-tagged LC3   (mTagRFP-mWasabi-LC3) reveals that high-dose rapamycin impairs   autophagic flux in cancer cells

doi:10.4161/auto.20284

CORC > 北京大学 > 化学与分子工程学院

	Monitoring autophagic flux by an improved tandem fluorescent-tagged LC3 (mTagRFP-mWasabi-LC3) reveals that high-dose rapamycin impairs autophagic flux in cancer cells
	Zhou, Cuihong ; Zhong, Wu ; Zhou, Jun ; Sheng, Fugeng ; Fang, Ziyuan ; Wei, Yue ; Chen, Yingyu ; Deng, Xiaoyan ; Xia, Bin ; Lin, Jian
刊名	autophagy
	2012
关键词	autophagy autophagic flux autophagosome autolysosome lysosome LC3 tandem fluorescent-tagged LC3 mRFP-EGFP-LC3 mTagRFP-mWasabi-LC3 rapamycin cisplatin staurosporine Z18
DOI	10.4161/auto.20284
英文摘要	Monitoring autophagic flux is important for the analysis of autophagy. Tandem fluorescent-tagged LC3 (mRFP-EGFP-LC3) is a convenient assay for monitoring autophagic flux based on different pH stability of EGFP and mRFP fluorescent proteins. However, it has been reported that there is still weak fluorescence of EGFP in acidic environments (pH between 4 and 5) or acidic lysosomes. So it is possible that autolysosomes are labeled with yellow signals (GFP(+)RFP(+) puncta), which results in misinterpreting autophagic flux results. Therefore, it is desirable to choose a monomeric green fluorescent protein that is more acid sensitive than EGFP in the assay of autophagic flux. Here, we report on an mTagRFP-mWasabi-LC3 reporter, in which mWasabi is more acid sensitive than EGFP and has no fluorescence in acidic lysosomes. Meanwhile, mTagRFP-mWasabi-LC3 Delta G was constructed as the negative control for this assay. Compared with mRFP-EGFP-LC3, our results showed that this reporter is more sensitive and accurate in detecting the accumulation of autophagosomes and autolysosomes. Using this reporter, we find that high-dose rapamycin (30 mu M) will impair autophagic flux, inducing many more autophagosomes than autolysosomes in HeLa cells, while low-dose rapamycin (500 nM) has an opposite effect. In addition, other chemical autophagy inducers (cisplatin, staurosporine and Z18) also elicit much more autophagosomes at high doses than those at low doses. Our results suggest that the dosage of chemical autophagy inducers would obviously influence autophagic flux in cells.; http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcApp=PARTNER_APP&SrcAuth=LinksAMR&KeyUT=WOS:000208891300006&DestLinkType=FullRecord&DestApp=ALL_WOS&UsrCustomerID=8e1609b174ce4e31116a60747a720701 ; Cell Biology; SCI(E); 61; ARTICLE; 8; 1215-1226; 8
语种	英语
内容类型	期刊论文
源URL	[http://ir.pku.edu.cn/handle/20.500.11897/150911]
专题	化学与分子工程学院
推荐引用方式 GB/T 7714	Zhou, Cuihong,Zhong, Wu,Zhou, Jun,et al. Monitoring autophagic flux by an improved tandem fluorescent-tagged LC3 (mTagRFP-mWasabi-LC3) reveals that high-dose rapamycin impairs autophagic flux in cancer cells[J]. autophagy,2012.
APA	Zhou, Cuihong.,Zhong, Wu.,Zhou, Jun.,Sheng, Fugeng.,Fang, Ziyuan.,...&Lin, Jian.(2012).Monitoring autophagic flux by an improved tandem fluorescent-tagged LC3 (mTagRFP-mWasabi-LC3) reveals that high-dose rapamycin impairs autophagic flux in cancer cells.autophagy.
MLA	Zhou, Cuihong,et al."Monitoring autophagic flux by an improved tandem fluorescent-tagged LC3 (mTagRFP-mWasabi-LC3) reveals that high-dose rapamycin impairs autophagic flux in cancer cells".autophagy (2012).