title: High-Throughput Quantification of GFP-LC3+Dots by Automated Fluorescence Microscopy.
abstract: Macroautophagy is a specific variant of autophagy that involves a dedicated double-membraned organelle commonly known as autophagosome. Various methods have been developed to quantify the size of the autophagosomal compartment, which is an indirect indicator of macroautophagic responses, based on the peculiar ability of microtubule-associated protein 1 light chain 3 beta (MAP1LC3B; best known as LC3) to accumulate in forming autophagosomes upon maturation. One particularly convenient method to monitor the accumulation of mature LC3 within autophagosomes relies on a green fluorescent protein (GFP)-tagged variant of this protein and fluorescence microscopy. In physiological conditions, cells transfected temporarily or stably with a GFP-LC3-encoding construct exhibit a diffuse green fluorescence over the cytoplasm and nucleus. Conversely, in response to macroautophagy-promoting stimuli, the GFP-LC3 signal becomes punctate and often (but not always) predominantly cytoplasmic. The accumulation of GFP-LC3 in cytoplasmic dots, however, also ensues the blockage of any of the steps that ensure the degradation of mature autophagosomes, calling for the implementation of strategies that accurately discriminate between an increase in autophagic flux and an arrest in autophagic degradation. Various cell lines have been engineered to stably express GFP-LC3, which-combined with the appropriate controls of flux, high-throughput imaging stations, and automated image analysis-offer a relatively straightforward tool to screen large chemical or biological libraries for inducers or inhibitors of autophagy. Here, we describe a simple and robust method for the high-throughput quantification of GFP-LC3+dots by automated fluorescence microscopy.
authors.affiliation: Gustave Roussy Cancer Campus, Villejuif, France; INSERM, U1138, Paris, France; Equipe 11 labellisée par la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France; Université Paris Descartes/Paris V, Sorbonne Paris Cité, Paris, France; Université Pierre et Marie Curie/Paris VI, Paris, France. Electronic address: chemabsp@gmail.com.
authors.forename: J M
authors.initials: JM
authors.lastname: Bravo-San Pedro
authors.affiliation: Gustave Roussy Cancer Campus, Villejuif, France; INSERM, U1138, Paris, France; Equipe 11 labellisée par la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France; Université Paris Descartes/Paris V, Sorbonne Paris Cité, Paris, France; Université Pierre et Marie Curie/Paris VI, Paris, France.
authors.forename: F
authors.initials: F
authors.lastname: Pietrocola
authors.affiliation: Gustave Roussy Cancer Campus, Villejuif, France; INSERM, U1138, Paris, France; Equipe 11 labellisée par la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France; Université Paris Descartes/Paris V, Sorbonne Paris Cité, Paris, France; Université Pierre et Marie Curie/Paris VI, Paris, France; Faculté de Medicine, Université Paris Saclay/Paris XI, Le Kremlin-Bicêtre, France.
authors.forename: V
authors.initials: V
authors.lastname: Sica
authors.affiliation: Gustave Roussy Cancer Campus, Villejuif, France; INSERM, U1138, Paris, France; Equipe 11 labellisée par la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France; Université Paris Descartes/Paris V, Sorbonne Paris Cité, Paris, France; Université Pierre et Marie Curie/Paris VI, Paris, France.
authors.forename: V
authors.initials: V
authors.lastname: Izzo
authors.affiliation: INSERM, U1138, Paris, France; Equipe 11 labellisée par la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France; Université Paris Descartes/Paris V, Sorbonne Paris Cité, Paris, France; Université Pierre et Marie Curie/Paris VI, Paris, France; Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France.
authors.forename: A
authors.initials: A
authors.lastname: Sauvat
authors.affiliation: INSERM, U1138, Paris, France; Equipe 11 labellisée par la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France; Université Paris Descartes/Paris V, Sorbonne Paris Cité, Paris, France; Université Pierre et Marie Curie/Paris VI, Paris, France; Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France.
authors.forename: O
authors.initials: O
authors.lastname: Kepp
authors.affiliation: Gustave Roussy Cancer Campus, Villejuif, France; INSERM, U1138, Paris, France; Equipe 11 labellisée par la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France; Université Paris Descartes/Paris V, Sorbonne Paris Cité, Paris, France; Université Pierre et Marie Curie/Paris VI, Paris, France.
authors.forename: M C
authors.initials: MC
authors.lastname: Maiuri
authors.affiliation: INSERM, U1138, Paris, France; Equipe 11 labellisée par la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France; Université Paris Descartes/Paris V, Sorbonne Paris Cité, Paris, France; Université Pierre et Marie Curie/Paris VI, Paris, France; Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France; Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP, Paris, France; Karolinska University Hospital, Stockholm, Sweden.
authors.forename: G
authors.initials: G
authors.lastname: Kroemer
authors.affiliation: Gustave Roussy Cancer Campus, Villejuif, France; INSERM, U1138, Paris, France; Equipe 11 labellisée par la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France; Université Paris Descartes/Paris V, Sorbonne Paris Cité, Paris, France; Université Pierre et Marie Curie/Paris VI, Paris, France; Weill Cornell Medical College, New York, NY, United States. Electronic address: deadoc@vodafone.it.
authors.forename: L
authors.initials: L
authors.lastname: Galluzzi
mesh_terms: D001331:Automation; D000071182:Autophagosomes / Q000378:metabolism*; D045744:Cell Line, Tumor; D003593:Cytoplasm / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D007091:Image Processing, Computer-Assisted; D008856:Microscopy, Fluorescence / Q000379:methods*; D008869:Microtubule-Associated Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D009369:Neoplasms / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism
chemical_list: C502070:MAP1LC3B protein, human; D008869:Microtubule-Associated Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins
keywords: Cancer; High-throughput screening; ImageXpress Micro XLS Widefield High-Content Analysis System; Lysosomal inhibitors; Nutrient deprivation; Rapamycin