Biomaterial Database

Monitoring autophagy in mammalian cultured cells through the dynamics of LC3. 2009

Shunsuke Kimura, and Naonobu Fujita, and Takeshi Noda, and Tamotsu Yoshimori

Department of Cellular Regulation, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.

In this chapter, we introduce several methods that rely on the analysis of LC3, a versatile marker protein of autophagic structures in mammalian cultured cells. The appearance of LC3-positive puncta is indicative of the induction of autophagy, and it is observed either by immunofluorescence or by GFP-based microscopy. The maturation process by which autophagosomes are converted into autolysosomes can be monitored by the GFP and RFP tandemly tagged LC3 (tfLC3) method. Lysosomal turnover of LC3 is a good index of the proceeding of autophagy and can be assessed by Western blotting. These methods will provide a relatively easy assessment of autophagy, and the details of the procedure will be described along with possible pitfalls.

UI	MeSH Term	Description	Entries
D008869	Microtubule-Associated Proteins	High molecular weight proteins found in the MICROTUBULES of the cytoskeletal system. Under certain conditions they are required for TUBULIN assembly into the microtubules and stabilize the assembled microtubules.	Ensconsin,Epithelial MAP, 115 kDa,Epithelial Microtubule-Associate Protein, 115 kDa,MAP4,Microtubule Associated Protein,Microtubule Associated Protein 4,Microtubule Associated Protein 7,Microtubule-Associated Protein,Microtubule-Associated Protein 7,E-MAP-115,MAP1 Microtubule-Associated Protein,MAP2 Microtubule-Associated Protein,MAP3 Microtubule-Associated Protein,Microtubule Associated Proteins,Microtubule-Associated Protein 1,Microtubule-Associated Protein 2,Microtubule-Associated Protein 3,7, Microtubule-Associated Protein,Associated Protein, Microtubule,E MAP 115,Epithelial Microtubule Associate Protein, 115 kDa,MAP1 Microtubule Associated Protein,MAP2 Microtubule Associated Protein,MAP3 Microtubule Associated Protein,Microtubule Associated Protein 1,Microtubule Associated Protein 2,Microtubule Associated Protein 3,Microtubule-Associated Protein, MAP1,Microtubule-Associated Protein, MAP2,Microtubule-Associated Protein, MAP3,Protein 7, Microtubule-Associated,Protein, Microtubule Associated,Protein, Microtubule-Associated
D002478	Cells, Cultured	Cells propagated in vitro in special media conducive to their growth. Cultured cells are used to study developmental, morphologic, metabolic, physiologic, and genetic processes, among others.	Cultured Cells,Cell, Cultured,Cultured Cell
D005455	Fluorescent Antibody Technique	Test for tissue antigen using either a direct method, by conjugation of antibody with fluorescent dye (FLUORESCENT ANTIBODY TECHNIQUE, DIRECT) or an indirect method, by formation of antigen-antibody complex which is then labeled with fluorescein-conjugated anti-immunoglobulin antibody (FLUORESCENT ANTIBODY TECHNIQUE, INDIRECT). The tissue is then examined by fluorescence microscopy.	Antinuclear Antibody Test, Fluorescent,Coon's Technique,Fluorescent Antinuclear Antibody Test,Fluorescent Protein Tracing,Immunofluorescence Technique,Coon's Technic,Fluorescent Antibody Technic,Immunofluorescence,Immunofluorescence Technic,Antibody Technic, Fluorescent,Antibody Technics, Fluorescent,Antibody Technique, Fluorescent,Antibody Techniques, Fluorescent,Coon Technic,Coon Technique,Coons Technic,Coons Technique,Fluorescent Antibody Technics,Fluorescent Antibody Techniques,Fluorescent Protein Tracings,Immunofluorescence Technics,Immunofluorescence Techniques,Protein Tracing, Fluorescent,Protein Tracings, Fluorescent,Technic, Coon's,Technic, Fluorescent Antibody,Technic, Immunofluorescence,Technics, Fluorescent Antibody,Technics, Immunofluorescence,Technique, Coon's,Technique, Fluorescent Antibody,Technique, Immunofluorescence,Techniques, Fluorescent Antibody,Techniques, Immunofluorescence,Tracing, Fluorescent Protein,Tracings, Fluorescent Protein
D006367	HeLa Cells	The first continuously cultured human malignant CELL LINE, derived from the cervical carcinoma of Henrietta Lacks. These cells are used for, among other things, VIRUS CULTIVATION and PRECLINICAL DRUG EVALUATION assays.	Cell, HeLa,Cells, HeLa,HeLa Cell
D006801	Humans	Members of the species Homo sapiens.	Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D001343	Autophagy	The segregation and degradation of various cytoplasmic constituents via engulfment by MULTIVESICULAR BODIES; VACUOLES; or AUTOPHAGOSOMES and their digestion by LYSOSOMES. It plays an important role in BIOLOGICAL METAMORPHOSIS and in the removal of bone by OSTEOCLASTS. Defective autophagy is associated with various diseases, including NEURODEGENERATIVE DISEASES and cancer.	Autophagocytosis,ER-Phagy,Lipophagy,Nucleophagy,Reticulophagy,Ribophagy,Autophagy, Cellular,Cellular Autophagy,ER Phagy
D049452	Green Fluorescent Proteins	Protein analogs and derivatives of the Aequorea victoria green fluorescent protein that emit light (FLUORESCENCE) when excited with ULTRAVIOLET RAYS. They are used in REPORTER GENES in doing GENETIC TECHNIQUES. Numerous mutants have been made to emit other colors or be sensitive to pH.	Green Fluorescent Protein,Green-Fluorescent Protein,Green-Fluorescent Proteins,Fluorescent Protein, Green,Fluorescent Proteins, Green,Protein, Green Fluorescent,Protein, Green-Fluorescent,Proteins, Green Fluorescent,Proteins, Green-Fluorescent