A new cell-based assay to evaluate myogenesis in mouse myoblast C2C12 cells

Kodaka, Manami; Yang, Zeyu; Nakagawa, Kentaro; Maruyama, Junichi; Xu, Xiaoyin; Sarkar, Aradhan; Ichimura, Ayana; Nasu, Yusuke; Ozawa, Toshio; Iwasa, Hiroaki; Ishigami‐Yuasa, Mari; Ito, Shigeru; Kagechika, Hiroyuki; Hata, Yoshinobu

doi:10.1016/j.yexcr.2015.06.015

Cited by 45 publications

(45 citation statements)

References 24 publications

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“…C2C12 is an immortalized murine myoblast cell line that has been extensively used to study regulation of myogenesis in vitro [ 35 – 37 ], and was chosen as a model system to express recombinant fluorescent proteins for the current study. C2C12 cells were transduced with lentiviruses encoding for fluorescent proteins localized to the cytoplasm and nucleus to create two combinations: (1) cytoplasmic GFP (cGFP)+nuclear mCherry (nmChery) and (2) cytoplasmic mCherry (cmCherry)+nuclear GFP (nGFP) ( Fig 1A ).…”

Section: Resultsmentioning

confidence: 99%

“…Several fluorescent tagging tools have been developed to monitor C2C12 cell line myoblast differentiation (see Table 2 for a comparison between such tools and the double label approach using C2C12 cells). For example, split GFP technology identifies fusion between cells from two populations expressing two complementary components of GFP, such that only fusion of cells from both populations results in fluorescence [ 35 ]. Alternatively, GFP expression can be driven by muscle creatine kinase (MCK) expressed during differentiation [ 54 ].…”

Section: Discussionmentioning

confidence: 99%

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A real-time monitoring platform of myogenesis regulators using double fluorescent labeling

Niu¹,

Zhou²,

Prim³

et al. 2018

PLoS ONE

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Real-time, quantitative measurement of muscle progenitor cell (myoblast) differentiation is an important tool for skeletal muscle research and identification of drugs that support skeletal muscle regeneration. While most quantitative tools rely on sacrificial approach, we developed a double fluorescent tagging approach, which allows for dynamic monitoring of myoblast differentiation through assessment of fusion index and nuclei count. Fluorescent tagging of both the cell cytoplasm and nucleus enables monitoring of cell fusion and the formation of new myotube fibers, similar to immunostaining results. This labeling approach allowed monitoring the effects of Myf5 overexpression, TNFα, and Wnt agonist on myoblast differentiation. It also enabled testing the effects of surface coating on the fusion levels of scaffold-seeded myoblasts. The double fluorescent labeling of myoblasts is a promising technique to visualize even minor changes in myogenesis of myoblasts in order to support applications such as tissue engineering and drug screening.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

A real-time monitoring platform of myogenesis regulators using double fluorescent labeling

Niu¹,

Zhou²,

Prim³

et al. 2018

PLoS ONE

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“…We then used 293T cells stably transfected with either one of the two complementary fragments of the green fluorescence protein (GFP) to analyze syncytia formation upon transient transfection with full-length syn1 and selected variants. Syncytia formation leads to reconstitution of a functional GFP, allowing easy monitoring of cellecell fusion by bimolecular fluorescence complementation [51] (described in detail in Ref. [52] and in the Material and Methods Section 'Cellecell fusion assay (293T cells)').…”

Section: Potential Break Points Of the N-helix In The Prefusion Formmentioning

confidence: 99%

“…Plasmids pQCXIP-GFP1-10 (Addgene plasmid #68715) and pQCXIP-BSR-GFP11 (Addgene plasmid #68716), encoding a GFP variant residues 1e155 (strands b1e10) and residues 156 to 172 (strand b11), respectively, were gifts from Yutaka Hata and are described in Ref. [51]. The synthetic gene encoding the full-length syn1 (residues 1e538, GenBank accession number Q9UQF0) was codon optimized for protein expression in mammalian cells, cloned into pcDNA3.1 expression vector, and purchased as such from GenScript.…”

Section: Plasmids and Cellsmentioning

confidence: 99%

X-ray Structures of the Post-fusion 6-Helix Bundle of the Human Syncytins and their Functional Implications

Ruigrok

Vaney

Buchrieser

et al. 2019

Journal of Molecular Biology

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The retroviral envelope-derived proteins syncytin-1 and syncytin-2 (syn1 and syn2) drive placentation in humans by forming a syncytiotophoblast, a structure allowing for an exchange interface between maternal and fetal blood during pregnancy. Despite their essential role, little is known about the molecular mechanism underlying the syncytins' function. We report here the X-ray structures of the syn1 and syn2 transmembrane subunit ectodomains, featuring a 6-helix bundle (6HB) typical of the post-fusion state of gamma-retrovirus and filovirus fusion proteins. Contrary to the filoviruses, for which the fusion glycoprotein was crystallized both in the post-fusion and in the spring-loaded pre-fusion form, the highly unstable nature of the syncytins' prefusion form has precluded structural studies. We undertook a proline-scanning approach searching for regions in the syn1 6HB central helix that tolerate the introduction of helix-breaker residues and still fold correctly in the pre-fusion form. We found that there is indeed such a region, located two a-helical turns downstream a stutter in the central coiled-coil helix -precisely where the breaks of the spring-loaded helix of the filoviruses map. These mutants were fusion-inactive as they cannot form the 6HB, similar to the "SOSIP" mutant of HIV Env that allowed the high-resolution structural characterization of its labile pre-fusion form.These results now open a new window of opportunity to engineer more stable variants of the elusive pre-fusion trimer of the syncytins and other gamma-retroviruses envelope proteins for structural characterization.

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“…The involvement and therapeutic potential of taurine have been discussed in pathophysiological conditions and skeletal muscle myopathy [83] . The screening systems to discover new therapeutic molecules and to evaluate compounds involved in satellite cell proliferation and fusion using human and mouse primary myoblast as well as mouse C2C12 cells are summarized in Table 1 [84] , [85] , [86] , [87] , [88] , [89] , [90] .…”

Section: Possible Therapeutic Intervention Strategiesmentioning

confidence: 99%

Molecular mechanisms and therapeutic interventions in sarcopenia

Park

Kwon

2017

Osteoporosis and Sarcopenia

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Sarcopenia is the degenerative loss of muscle mass and function with aging. Recently sarcopenia was recognized as a clinical disease by the International Classification of Disease, 10th revision, Clinical Modification. An imbalance between protein synthesis and degradation causes a gradual loss of muscle mass, resulting in a decline of muscle function as a progress of sarcopenia. Many mechanisms involved in the onset of sarcopenia include age-related factors as well as activity-, disease-, and nutrition-related factors. The stage of sarcopenia reflecting the severity of conditions assists clinical management of sarcopenia. It is important that systemic descriptions of the disease conditions include age, sex, and other environmental risk factors as well as levels of physical function. To develop a new therapeutic intervention needed is the detailed understanding of molecular and cellular mechanisms by which apoptosis, autophagy, atrophy, and hypertrophy occur in the muscle stem cells, myotubes, and/or neuromuscular junction. The new strategy to managing sarcopenia will be signal-modulating small molecules, natural compounds, repurposing of old drugs, and muscle-specific microRNAs.

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A new cell-based assay to evaluate myogenesis in mouse myoblast C2C12 cells

Cited by 45 publications

References 24 publications

A real-time monitoring platform of myogenesis regulators using double fluorescent labeling

A real-time monitoring platform of myogenesis regulators using double fluorescent labeling

X-ray Structures of the Post-fusion 6-Helix Bundle of the Human Syncytins and their Functional Implications

Molecular mechanisms and therapeutic interventions in sarcopenia

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