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A rapid and high content assay that measures cyto-ID-stained autophagic compartments and estimates autophagy flux with potential clinical applications

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dc.contributor.authorGuo, Sujuanen
dc.contributor.authorLiang, Yanpingen
dc.contributor.authorMurphy, Susan F.en
dc.contributor.authorHuang, Angelaen
dc.contributor.authorShen, Haihongen
dc.contributor.authorKelly, Deborah F.en
dc.contributor.authorSobrado, Pabloen
dc.contributor.authorSheng, Zhien
dc.contributor.departmentBiochemistryen
dc.contributor.departmentBiological Sciencesen
dc.contributor.departmentBiomedical Sciences and Pathobiologyen
dc.contributor.departmentPopulation Health Sciencesen
dc.contributor.departmentFralin Biomedical Research Instituteen
dc.contributor.departmentFralin Life Sciences Instituteen
dc.contributor.departmentCenter for Drug Discoveryen
dc.date.accessioned2017-03-30T18:00:42Zen
dc.date.available2017-03-30T18:00:42Zen
dc.date.issued2015-03-01en
dc.description.abstractThe lack of a rapid and quantitative autophagy assay has substantially hindered the development and implementation of autophagy-targeting therapies for a variety of human diseases. To address this critical issue, we developed a novel autophagy assay using the newly developed Cyto-ID fluorescence dye. We first verified that the Cyto-ID dye specifically labels autophagic compartments with minimal staining of lysosomes and endosomes. We then developed a new Cyto-ID fluorescence spectrophotometric assay that makes it possible to estimate autophagy flux based on measurements of the Cyto-ID-stained autophagic compartments. By comparing to traditional autophagy approaches, we found that this assay yielded a more sensitive, yet less variable, quantification of the stained autophagic compartments and the estimate of autophagy flux. Furthermore, we tested the potential application of this autophagy assay in high throughput research by integrating it into an RNA interference (RNAi) screen and a small molecule screen. The RNAi screen revealed WNK2 and MAP3K6 as autophagy-modulating genes, both of which inhibited the MTOR pathway. Similarly, the small molecule screen identified sanguinarine and actinomycin D as potent autophagy inducers in leukemic cells. Moreover, we successfully detected autophagy responses to kinase inhibitors and chloroquine in normal or leukemic mice using this assay. Collectively, this new Cyto-ID fluorescence spectrophotometric assay provides a rapid, reliable quantification of autophagic compartments and estimation of autophagy flux with potential applications in developing autophagy-related therapies and as a test to monitor autophagy responses in patients being treated with autophagy-modulating drugs.en
dc.description.versionPublished versionen
dc.format.extent560 - 572 (13) page(s)en
dc.format.mimetypeapplication/pdfen
dc.identifier.doihttps://doi.org/10.1080/15548627.2015.1017181en
dc.identifier.issn1554-8627en
dc.identifier.issue3en
dc.identifier.orcidSobrado, P [0000-0003-1494-5382]en
dc.identifier.urihttp://hdl.handle.net/10919/76732en
dc.identifier.volume11en
dc.language.isoenen
dc.publisherTaylor & Francisen
dc.relation.urihttp://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcApp=PARTNER_APP&SrcAuth=LinksAMR&KeyUT=WOS:000353587600012&DestLinkType=FullRecord&DestApp=ALL_WOS&UsrCustomerID=930d57c9ac61a043676db62af60056c1en
dc.rightsCreative Commons Attribution-NonCommercial 3.0 Unporteden
dc.rights.holderThe Author(s)en
dc.rights.urihttp://creativecommons.org/licenses/by-nc/3.0/en
dc.subjectCell Biologyen
dc.subjectautophagyen
dc.subjectautophagy fluxen
dc.subjectautophagy responseen
dc.subjectCyto-IDen
dc.subjectRNA interference screenen
dc.subjectsmall molecule screenen
dc.subjectspectrophotometric assayen
dc.subject3-MAen
dc.subject3-methyladenineen
dc.subjectFBSen
dc.subjectfetal bovine serumen
dc.subjectGFPen
dc.subjectgreen fluorescent proteinen
dc.subjectLAMP1en
dc.subjectlysosomal-associated membrane protein 1en
dc.subjectMAP1LC3Ben
dc.subjectLC3Ben
dc.subjectmicrotubule-associated protein 1 light chain 3 betaen
dc.subjectMAP3K6en
dc.subjectmitogen-activated protein kinase kinase kinase 6en
dc.subjectMDCen
dc.subjectmonodansylcadaverineen
dc.subjectmRFPen
dc.subjectmonomeric red fluorescent proteinen
dc.subjectMTORen
dc.subjectmechanistic target of rapamycinen
dc.subjectNSen
dc.subjectnonsilencingen
dc.subjectRAB5Aen
dc.subjectmember RAS oncogene familyen
dc.subjectRNAien
dc.subjectRNA interferenceen
dc.subjectshRNAen
dc.subjectshort-hairpin RNAen
dc.subjectSQSTM1en
dc.subjectsequestosome 1en
dc.subjectWNK2en
dc.subjectWNK lysine deficient protein kinase 2en
dc.subjectSIRNA SCREENen
dc.subjectLIVING CELLSen
dc.subjectTRANSCRIPTIONen
dc.subjectCHLOROQUINEen
dc.subjectKINASESen
dc.subjectDISEASEen
dc.subjectMARKERen
dc.subjectTRIALen
dc.subjectLC3en
dc.titleA rapid and high content assay that measures cyto-ID-stained autophagic compartments and estimates autophagy flux with potential clinical applicationsen
dc.title.serialAutophagyen
dc.typeArticle - Refereeden
dc.type.dcmitypeTexten
pubs.organisational-group/Virginia Techen
pubs.organisational-group/Virginia Tech/Agriculture & Life Sciencesen
pubs.organisational-group/Virginia Tech/Agriculture & Life Sciences/Biochemistryen
pubs.organisational-group/Virginia Tech/Agriculture & Life Sciences/CALS T&R Facultyen
pubs.organisational-group/Virginia Tech/All T&R Facultyen
pubs.organisational-group/Virginia Tech/Faculty of Health Sciencesen
pubs.organisational-group/Virginia Tech/University Research Institutesen
pubs.organisational-group/Virginia Tech/University Research Institutes/Virginia Tech Carilion Research Instituteen

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