Assays to Monitor Autophagy Progression in Cell Cultures

The vast number of implications of autophagy in multiple areas of life sciences and medicine has attracted the interest of numerous scientists that aim to unveil the role of this process in specific physiological and pathological contexts. Cell cultures are one of the most frequently used experimental setup for the investigation of autophagy. As a result, it is essential to assess this highly regulated molecular pathway with efficient and reliable methods. Each method has its own advantages and disadvantages. Here, we present a review summarizing the most established assays used to monitor autophagy induction and progression in cell cultures, in order to guide researchers in the selection of the most optimal solution for their experimental setup and design.

• Publication year

  2017

• Venue

  Cells

• Publication date

  2017-07-07

• Fields of study

  Biology, Medicine

• Identifiers
  DOI 10.3390/cells6030020PMID 28686195PMCID 5617966
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

• Posttranslational Modification

  2019cited by this paper
• F1000Prime recommendation of Structure of the human mTOR complex I and its implications for rapamycin inhibition.

  2018cited by this paper
• Turnover of Lipidated LC3 and Autophagic Cargoes in Mammalian Cells.

  2017cited by this paper
• Nonradioactive quantification of autophagic protein degradation with L-azidohomoalanine labeling

  2017cited by this paper
• A Simple Cargo Sequestration Assay for Quantitative Measurement of Nonselective Autophagy in Cultured Cells.

  2017cited by this paper
• Measurement of Autophagy by Flow Cytometry.

  2017cited by this paper
• PI3K-Akt-mTOR axis sustains rotavirus infection via the 4E-BP1 mediated autophagy pathway and represents an antiviral target

  2017cited by this paper
• Quantifying autophagy: Measuring LC3 puncta and autolysosome formation in cells using multispectral imaging flow cytometry.

  2017cited by this paper
• Ultrastructural Characterization of Phagophores Using Electron Tomography on Cryoimmobilized and Freeze Substituted Samples.

  2017cited by this paper
• Long-Lived Protein Degradation During Autophagy.

  2017cited by this paper
• Assessment of Posttranslational Modifications of ATG proteins.

  2017cited by this paper
• Renilla Luciferase-LC3 Based Reporter Assay for Measuring Autophagic Flux.

  2017cited by this paper
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  2017cited by this paper
• Visualization of reticulophagy in living cells using an endoplasmic reticulum-targeted p62 mutant

  2017cited by this paper
• Atg8 family LC3/GABARAP proteins are crucial for autophagosome–lysosome fusion but not autophagosome formation during PINK1/Parkin mitophagy and starvation

  2016cited by this paper
• Fine-tuning autophagy: from transcriptional to posttranslational regulation.

  2016cited by this paper
• Regulation of mATG9 trafficking by Src- and ULK1-mediated phosphorylation in basal and starvation-induced autophagy

  2016cited by this paper
• Insights into autophagosome biogenesis from in vitro reconstitutions

  2016cited by this paper
• An Autophagic Flux Probe that Releases an Internal Control.

  2016cited by this paper
• Autophagy: machinery and regulation

  2016cited by this paper
• ATG proteins: Are we always looking at autophagy?

  2016cited by this paper
• An siRNA screen for ATG protein depletion reveals the extent of the unconventional functions of the autophagy proteome in virus replication

  2016cited by this paper
• RCAD/Ufl1, a Ufm1 E3 ligase, is essential for hematopoietic stem cell function and murine hematopoiesis

  2015cited by this paper
• Function of human WIPI proteins in autophagosomal rejuvenation of endomembranes?

  2015cited by this paper
• Macroautophagic cargo sequestration assays.

  2015cited by this paper
• Posttranslational modification of autophagy-related proteins in macroautophagy

  2015cited by this paper
• A rapid and high content assay that measures cyto-ID-stained autophagic compartments and estimates autophagy flux with potential clinical applications

  2015cited by this paper
• Deacetylation of nuclear LC3 drives autophagy initiation under starvation.

  2015cited by this paper
• Assessing mammalian autophagy.

  2015cited by this paper
• Flow cytometric assays for the study of autophagy.

  2015cited by this paper
• Macroautophagy is dispensable for growth of KRAS mutant tumors and chloroquine efficacy

  2015cited by this paper
• Selective autophagy: xenophagy.

  2015cited by this paper
• Structured illumination microscopy and correlative microscopy to study autophagy.

  2015cited by this paper
• Selective autophagy: lysophagy.

  2015cited by this paper
• Autophagic bulk sequestration of cytosolic cargo is independent of LC3, but requires GABARAPs.

  2015cited by this paper
• Small Molecule Inhibition of the Autophagy Kinase ULK1 and Identification of ULK1 Substrates.

  2015cited by this paper
• Expression of the autophagy substrate SQSTM1/p62 is restored during prolonged starvation depending on transcriptional upregulation and autophagy-derived amino acids

  2014cited by this paper
• Development of a novel method for quantification of autophagic protein degradation by AHA labeling

  2014cited by this paper
• Novel Quantitative Autophagy Analysis by Organelle Flow Cytometry after Cell Sonication

  2014cited by this paper
• Caloric restriction mimetics: natural/physiological pharmacological autophagy inducers

  2014cited by this paper
• Emerging role of selective autophagy in human diseases

  2014cited by this paper
• Imaging endosomes and autophagosomes in whole mammalian cells using correlative cryo-fluorescence and cryo-soft X-ray microscopy (cryo-CLXM)☆

  2014cited by this paper
• Autophagy and human diseases

  2013cited by this paper
• Non-autophagic roles of autophagy-related proteins

  2013cited by this paper
• Differential regulation of distinct Vps34 complexes by AMPK in nutrient stress and autophagy.

  2013cited by this paper
• The emerging links between sirtuins and autophagy.

  2013cited by this paper
• Emerging regulation and functions of autophagy

  2013cited by this paper
• Hidden Behind Autophagy: The Unconventional Roles of ATG Proteins

  2013cited by this paper
• Cutting a fine figure

  2013cited by this paper
• Tools and techniques to measure mitophagy using fluorescence microscopy

  2013cited by this paper
• A current perspective of autophagosome biogenesis

  2013cited by this paper
• Guidelines for the use and interpretation of assays for monitoring autophagy

  2012influential reference
• Selective Types of Autophagy

  2012cited by this paper
• Pro-autophagic polyphenols reduce the acetylation of cytoplasmic proteins

  2012cited by this paper
• A novel image-based cytometry method for autophagy detection in living cells

  2012cited by this paper
• Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

  2012cited by this paper
• Autophagy: More Than a Nonselective Pathway

  2012cited by this paper
• p62, Ref(2)P and ubiquitinated proteins are conserved markers of neuronal aging, aggregate formation and progressive autophagic defects

  2011cited by this paper
• The serine/threonine kinase ULK1 is a target of multiple phosphorylation events.

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• Lanatoside C sensitizes glioblastoma cells to tumor necrosis factor-related apoptosis-inducing ligand and induces an alternative cell death pathway.

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• Biogenesis and cargo selectivity of autophagosomes.

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• Seeing is believing: The impact of electron microscopy on autophagy research

  2011cited by this paper
• Hsp90-Cdc37 chaperone complex regulates Ulk1- and Atg13-mediated mitophagy.

  2011cited by this paper
• Double membranes vs. lipid bilayers, and their significance for correct identification of macroautophagic structures

  2011cited by this paper
• AMP-activated protein kinase: nature's energy sensor.

  2011cited by this paper
• Structure of the human mTOR complex I and its implications for rapamycin inhibition.

  2010cited by this paper
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  2010cited by this paper
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  2010cited by this paper
• LC3 and GATE‐16/GABARAP subfamilies are both essential yet act differently in autophagosome biogenesis

  2010cited by this paper
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  2010cited by this paper
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  2009cited by this paper
• A role for NBR1 in autophagosomal degradation of ubiquitinated substrates.

  2009cited by this paper
• Assaying of autophagic protein degradation.

  2009cited by this paper
• The late stages of autophagy: how does the end begin?

  2009cited by this paper
• To be or not to be? Examples of incorrect identification of autophagic compartments in conventional transmission electron microscopy of mammalian cells

  2008cited by this paper
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  2008cited by this paper
• Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum

  2008cited by this paper
• In Brief

  2007cited by this paper
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  2006cited by this paper
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  2006cited by this paper
• p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death

  2005cited by this paper
• Analyses of Galpha-interacting protein and activator of G-protein-signaling-3 functions in macroautophagy.

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  2003cited by this paper
• Changes in the proteolytic activities of proteasomes and lysosomes in human fibroblasts produced by serum withdrawal, amino-acid deprivation and confluent conditions.

  2003cited by this paper
• LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing

  2000influential reference
• Bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase, inhibits acidification and protein degradation in lysosomes of cultured cells.

  1991cited by this paper
• Nonselective autophagy of cytosolic enzymes by isolated rat hepatocytes

  1990cited by this paper
• Inhibition of the lysosomal pathway of protein degradation in isolated rat hepatocytes by ammonia, methylamine, chloroquine and leupeptin.

  1979cited by this paper
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  1967cited by this paper
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  year unknowncited by this paper

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