Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)¹

Abstract

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for <i>bona fide</i> autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Keywords

Affiliated Institutions

• University of Dundee GB
• The Wistar Institute US
• Telethon Institute Of Genetics And Medicine IT
• Università di Camerino IT
• University of Alabama at Birmingham US
• Koç University TR
• Cologne Excellence Cluster on Cellular Stress Responses in Aging Associated Diseases DE
• Universidad de La Laguna ES
• University of California, Davis US
• Instituto Biofisika ES
• National Center of Neurology and Psychiatry JP
• MRC Protein Phosphorylation and Ubiquitylation Unit GB
• Medical University of Graz AT
• University of Copenhagen DK
• INCLIVA Health Research Institute ES
• City of Hope US
• University of Bologna IT
• VIB-KU Leuven Center for Cancer Biology BE
• Institute of Neurobiology BG
• Universidad de Buenos Aires AR
• Imperial College London GB
• Infectious Disease Research Institute US
• The University of Sydney AU
• University of Crete GR
• Centre Méditerranéen de Médecine Moléculaire FR
• Centro de Investigación Biomédica en Red de Fragilidad y Envejecimiento Saludable ES
• i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto PT
• Leibniz Institute of Plant Biochemistry DE
• Hospital Universitario de Canarias ES
• Institute of Cancer Research GB
• Nihon University JP
• University of Manitoba CA
• Aarhus University DK
• The Francis Crick Institute GB
• McGill University Health Centre CA
• Consejo Nacional de Investigaciones Científicas y Técnicas AR
• Center for Systems Biology Dresden DE
• Rede de Química e Tecnologia PT
• University of Rajshahi BD
• Cleveland Clinic US
• Universidade do Porto PT
• Consejo Superior de Investigaciones Científicas ES
• Centro de Investigación Biomédica en Red ES
• University of Maryland, Baltimore US
• Medical University of Vienna AT
• University of Calabria IT
• Centre de Recherches sur la Cognition Animale FR
• Stanford University US
• Sapienza University of Rome IT
• Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas ES
• University of Michigan US
• Ain Shams University EG
• University of California, San Francisco US
• Tohoku University JP
• Cedars-Sinai Smidt Heart Institute US
• Ben-Gurion University of the Negev IL
• Robert Bosch (Australia) AU
• Universidade Nove de Julho BR
• Universidad de Sevilla ES
• Johns Hopkins Medicine US
• Northeast Ohio Medical University US
• Newcastle University GB
• Universitat de València ES
• Danish Cancer Society DK
• University of Toronto CA
• Polish Academy of Sciences PL
• Jikei University School of Medicine JP
• Beckman Research Institute
• University of Cologne DE
• Colby College US
• Institut d'Investigació Biomédica de Bellvitge ES
• Cedars-Sinai Medical Center US
• Bulgarian Academy of Sciences BG
• Birmingham VA Medical Center US
• University of Palermo IT
• Centre National de la Recherche Scientifique FR
• Universidade Nova de Lisboa PT
• European Institute of Oncology IT
• UiT The Arctic University of Norway
• Universidade Federal de Viçosa BR
• University of Helsinki FI
• Meir Medical Center IL
• Albert Einstein College of Medicine US
• Universitat de Barcelona ES
• University of Pavia IT
• Pasteur Institute of Iran IR
• Université Toulouse III - Paul Sabatier FR
• Islamic University of Gaza PS
• York College, City University of New York US
• National Eye Institute US
• Instituto Cajal ES
• Universidad Mayor CL
• Howard University US
• Children's Hospital of Pittsburgh US
• University of Tennessee at Knoxville US
• Innsbruck Medical University AT
• The University of Texas Medical Branch at Galveston US
• Lund University SE
• Indian Institute of Technology Ropar IN
• Max Planck Institute for Biology of Ageing DE
• Tokyo University of Pharmacy and Life Sciences JP
• University of Bristol GB
• National Institutes of Health US
• Universidad Nacional de Córdoba AR
• University of Pittsburgh Medical Center US
• University of the Basque Country ES
• Tel Aviv University IL
• Inserm FR
• Gulf Medical University AE
• Iowa State University US
• Universidade Federal de São Paulo BR
• Johannes Gutenberg University Mainz DE
• National Heart Lung and Blood Institute US
• Sabancı Üniversitesi TR
• Johns Hopkins University US
• Inflammation Research Foundation US
• The University of Osaka JP
• Hebrew University of Jerusalem IL

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