title: A method for the establishment of a cell line with stable expression             of the GFP-LC3 reporter protein.
abstract: As the function of autophagy becomes evident in a number of diseases, including             cancer and infection, it is crucial to construct macrophage cell lines with stable             expression of the microtubule-associated protein light chain 3 (GFP-LC3). In this             study, a mouse LC3 open-reading frame was amplified by RT-PCR, and cloned into             the pEGFP-C1 plasmid for expression of the GFP-LC3 fusion protein. The recombinant             plasmid was transfected into RAW264.7 cells using Lipofectamine 2000 reagent and             stably transfected clones were selected by G418 screening. Autophagic puncta formation             was observed by fluorescense microscopy. Additionally, we found that starvation             treatment induced a significant increase in the number of autophagosomes, while             wortmannin treatment significantly repressed the formation of autophagosomes.             This study indicated that the RAW264.7 cell line stably expressing GFP-LC3 is             available for use in a GFP-LC3 puncta formation assay, and may contribute to basic             investigations of autophagic function or drug screening targeted at autophagy.
authors: Department of Medical Immunology, Medical School, Anhui University of Science and Technology, Huainan, Anhui 232001, PR China.
authors: Dong
authors: D
authors: Hu
authors: Jing
authors: J
authors: Wu
authors: Lifa
authors: L
authors: Xu
authors: Rongbo
authors: R
authors: Zhang
authors: Liping
authors: L
authors: Chen
mesh_terms: D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D000935:Antifungal Agents / Q000494:pharmacology; D001343:Autophagy / Q000187:drug effects; D002460:Cell Line; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D008055:Lipids / Q000737:chemistry; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D000077191:Wortmannin
chemical_list: D000730:Androstadienes; D000935:Antifungal Agents; D008055:Lipids; C086724:Lipofectamine; C480860:Map1lc3b protein, mouse; D008869:Microtubule-Associated Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D000077191:Wortmannin