title: A new cell-based assay to evaluate myogenesis in mouse myoblast C2C12 cells.
abstract: The development of the efficient screening system of detecting compounds that promote myogenesis and prevent muscle atrophy is important. Mouse C2C12 cells are widely used to evaluate myogenesis but the procedures of the assay are not simple and the quantification is not easy. We established C2C12 cells expressing the N-terminal green fluorescence protein (GFP) and the C-terminal GFP (GFP1-10 and GFP11 cells). GFP1-10 and GFP11 cells do not exhibit GFP signals until they are fused. The signal intensity correlates with the expression of myogenic markers and myofusion. Myogenesis-promoting reagents, such as insulin-like growth factor-1 (IGF1) and β-guanidinopropionic acid (GPA), enhance the signals, whereas the poly-caspase inhibitor, z-VAD-FMK, suppresses it. GFP signals are observed when myotubes formed by GFP1-10 cells are fused with single nuclear GFP11 cells, and enhanced by IGF1, GPA, and IBS008738, a recently-reported myogenesis-promoting reagent. Fusion between myotubes formed by GFP1-10 and GFP11 cells is associated with the appearance of GFP signals. IGF1 and GPA augment these signals, whereas NSC23766, Rac inhibitor, decreases them. The conditioned medium of cancer cells suppresses GFP signals during myogenesis and reduces the width of GFP-positive myotubes after differentiation. Thus the novel split GFP-based assay will provide the useful method for the study of myogenesis, myofusion, and atrophy.
authors.affiliation: Department of Medical Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan.
authors.forename: Manami
authors.initials: M
authors.lastname: Kodaka
authors.affiliation: Department of Medical Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan; Department of Ultrasound, Shengjing Hospital of China Medical University, Shenyang, China.
authors.forename: Zeyu
authors.initials: Z
authors.lastname: Yang
authors.affiliation: Department of Medical Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan.
authors.forename: Kentaro
authors.initials: K
authors.lastname: Nakagawa
authors.affiliation: Department of Medical Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan.
authors.forename: Junichi
authors.initials: J
authors.lastname: Maruyama
authors.affiliation: Department of Medical Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan; Department of Breast Surgery, The Second Affiliated Hospital of Wenzhou Medical University, Wenzhou, China.
authors.forename: Xiaoyin
authors.initials: X
authors.lastname: Xu
authors.affiliation: Department of Medical Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan.
authors.forename: Aradhan
authors.initials: A
authors.lastname: Sarkar
authors.affiliation: Department of Medical Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan.
authors.forename: Ayana
authors.initials: A
authors.lastname: Ichimura
authors.affiliation: Department of Breast Surgery, The Second Affiliated Hospital of Wenzhou Medical University, Wenzhou, China.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Nasu
authors.affiliation: Department of Chemistry, School of Science, The University of Tokyo, Tokyo, Japan.
authors.forename: Takeaki
authors.initials: T
authors.lastname: Ozawa
authors.affiliation: Department of Medical Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Iwasa
authors.affiliation: Chemical Biology Screening Center, Tokyo Medical and Dental University, Tokyo, Japan.
authors.forename: Mari
authors.initials: M
authors.lastname: Ishigami-Yuasa
authors.affiliation: Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo, Japan.
authors.forename: Shigeru
authors.initials: S
authors.lastname: Ito
authors.affiliation: Chemical Biology Screening Center, Tokyo Medical and Dental University, Tokyo, Japan; Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Kagechika
authors.affiliation: Department of Medical Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan; Center for Brain Integration Research, Tokyo Medical and Dental University, Tokyo, Japan. Electronic address: yuhammch@tmd.ac.jp.
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Hata
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000634:Aminoquinolines / Q000494:pharmacology; D000818:Animals; D061945:Caspase Inhibitors / Q000494:pharmacology; D002454:Cell Differentiation; D002459:Cell Fusion; D002460:Cell Line; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006146:Guanidines / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D051379:Mice; D024510:Muscle Development / Q000187:drug effects*; D018485:Muscle Fibers, Skeletal / Q000166:cytology*; D009133:Muscular Atrophy / Q000517:prevention & control*; D032446:Myoblasts / Q000166:cytology*; D011422:Propionates / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D020744:rac GTP-Binding Proteins / Q000037:antagonists & inhibitors
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D000634:Aminoquinolines; D061945:Caspase Inhibitors; D006146:Guanidines; C000588695:IBS008738; D007093:Imidazoles; C487513:NSC 23766; D011422:Propionates; D011743:Pyrimidines; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C519530:insulin-like growth factor-1, mouse; D049452:Green Fluorescent Proteins; D007334:Insulin-Like Growth Factor I; D020744:rac GTP-Binding Proteins; C005775:guanidinopropionic acid
keywords: C2C12; Cachexia; GFP; Myofusion; Myogenesis