【摘 要】
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The activation of autophagy was commonly detected by the formation of autophagosome,or the conversion of LC3 from Ⅰ to Ⅱ forms,accompanied with the increased levels of autophagy-related proteins.Nonet
【机 构】
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Department of Biochemistry and Molecular Biology,School of Basic Medical Sciences,Capital Medical Un
【出 处】
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中国生物化学与分子生物学会2016年全国学术会议
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The activation of autophagy was commonly detected by the formation of autophagosome,or the conversion of LC3 from Ⅰ to Ⅱ forms,accompanied with the increased levels of autophagy-related proteins.Nonetheless,the assays to allow quantifiable measures of the dynamics of cellular autophagy,which was referred as the “autophagic flux”,remained to be scarce.In the present study,we discovered a mutant of p62/SQSTM1 lacking a UBA(ubiquitin binding domain,p62ΔU)was able to escape from autophagic degradation,unlike the wild type proteins.By fusing wt-p62 or p62ΔU with luciferase,naming Luc2p-p62 or Luc2p-p62 p62ΔU,respectively,we developed a luciferase reporter system for the quantitative measurements of autophagic flux in cultured cells.U87 MG or U251 MG glioma cells ectopically expressing Luc2p-p62 or Luc2p-p62ΔU following transfection were indexed by the ratio of the accorded luminescence values,which indicated the dynamics of the autophagic flux.With this system,induction of autophagy via starvation and rapamycin greatly increased the ratio of Luc2p-p62/Luc2p-p62ΔU,which was drastically attenuated upon chloroquine treatment or knockdown of ATG5.The autophagic flux determined by this method was in well consistency with that reflected by the formation of autophagosome formation,p62/SQSTM1 protein degradation and LC3I/LC3II conversion,while manifesting much more convenience for examination and quantification.Furthermore,autophagic flux trigged with temozolomide(TMZ)was successfully determined in glioma cells with this novel monitoring system,showing its clinical potency.Our study provides a powerful tool for both the mechanistic and clinical researches of autophagy.
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