A new cell-based assay to evaluate myogenesis in mouse myoblast C2C12 cells
A new cell-based assay to evaluate myogenesis in mouse myoblast C2C12 cells
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DOI：
10.1016/j.yexcr.2015.06.015
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发表时间：
2015-08-15
影响因子：
3.7
通讯作者：
Hata, Yutaka
中科院分区：
文献类型：
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作者：
Kodaka, Manami;Yang, Zeyu;Hata, Yutaka
开发检测促进肌生成和防止肌肉萎缩的化合物的有效筛选系统是重要的。小鼠C2 C12细胞被广泛用于评价肌细胞发生，但其方法不简单，且不易定量。我们建立了表达N-末端绿色荧光蛋白（GFP）和C-末端GFP的C2 C12细胞（GFP 1 -10和GFP 11细胞）。GFP 1 -10和GFP 11细胞在融合之前不显示GFP信号。信号强度与肌源性标志物的表达和肌融合相关。肌生成促进试剂，如胰岛素样生长因子-1（IGF 1）和β-胍基丙酸（GPA），增强信号，而聚半胱天冬酶抑制剂z-VAD-FMK，抑制它。当GFP 1 -10细胞形成的肌管与单核GFP 11细胞融合时，观察到GFP信号，并被IGF 1，GPA和185008738增强，一种新近报道的促肌生成试剂。GFP 1 -10和GFP 11细胞形成的肌管之间的融合与GFP信号的出现有关。IGF 1和CPA增强这些信号，而Rac抑制剂NSC 23766降低它们。癌细胞的条件培养基在肌生成期间抑制GFP信号，并且在分化后减小GFP阳性肌管的宽度。因此，新的分裂GFP为基础的分析将提供有用的方法，研究肌发生，肌融合，萎缩。(C)2015 Elsevier Inc. All rights reserved.
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 beta-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 185008738, 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 CPA 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. (C) 2015 Elsevier Inc. All rights reserved.