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myoblast transfection

Moon Hee Lee, Min Ho Han, Dae-Sung Lee, Cheol Park, Su-Hyun Hong, Gi-Young Kim, Sang Hoon Hong, Kyoung Seob Song, Il-Whan Choi, Hee-Jae Cha, Yung Hyun Choi
In the present study, we investigated the cytoprotective efficacy of morin, a natural flavonoid, against oxidative stress and elucidated the underlying mechanisms in C2C12 myoblasts. Our results indicated that morin treatment prior to hydrogen peroxide (H2O2) exposure significantly increased cell viability and prevented the generation of reactive oxygen species. H2O2-induced comet-like DNA formation and γH2AX phosphorylation were also markedly suppressed by morin with a parallel inhibition of apoptosis in C2C12 myoblasts, suggesting that morin prevented H2O2-induced cellular DNA damage...
February 2017: International Journal of Molecular Medicine
Yonghua Liu, Weijie Wu, Huiguang Yang, Zhengming Zhou, Xiaojian Zhu, Chi Sun, Yuxi Liu, Zhaohui Yu, Yuyan Chen, Youhua Wang
Tripartite motif containing 32 (TRIM32), a member of the tripartite motif (TRIM) family, plays an indispensable role in myoblast proliferation. It also regulates neuron and skeletal muscle stem cell differentiation. Although it is of great importance, we know little about the roles of TRIM32 during peripheral nervous system injury. Here, we examined the dynamic changes of TRIM32 in acute sciatic nerve crush (SNC) model. After crush, TRIM32 rapidly increased and reached the climax at 1 week but then gradually declined to the normal level at 4 weeks post-injury...
December 26, 2016: Neurochemical Research
Kenneth C Ehrlich, Heather L Paterson, Michelle Lacey, Melanie Ehrlich
Tissue-specific enhancers are critical for gene regulation. In this study, we help elucidate the contribution of muscle-associated differential DNA methylation to the enhancer activity of highly muscle-specific genes. By bioinformatic analysis of 44 muscle-associated genes, we show that preferential gene expression in skeletal muscle (SkM) correlates with SkM-specific intragenic and intergenic enhancer chromatin and overlapping foci of DNA hypomethylation. Some genes, e.g., CASQ1 and FBXO32, displayed broad regions of both SkM- and heart-specific enhancer chromatin but exhibited focal SkM-specific DNA hypomethylation...
December 2016: Yale Journal of Biology and Medicine
Arnold Junior Tatsinkam, Naomi Rune, Joy Smith, Jill T Norman, Barbara Mulloy, Christopher C Rider
Gremlin-1, a bone morphogenetic protein (BMP) antagonist, has essential roles in kidney and limb bone development, and is important in chronic diseases including tissue fibrosis. It also functions as an activating ligand of the vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2), and binds strongly to the sulfated polysaccharide, heparin. Here we investigated the extent to which gremlin binds to the related polysaccharide heparan sulfate (HS), which unlike heparin is widely distributed spread within tissues...
December 12, 2016: International Journal of Biochemistry & Cell Biology
V K Chaithanya Ponnaluri, Kenneth C Ehrlich, Guoqiang Zhang, Michelle Lacey, Douglas Johnston, Sriharsa Pradhan, Melanie Ehrlich
Differentially methylated or hydroxymethylated regions (DMRs) in mammalian DNA are often associated with tissue-specific gene expression but the functional relationships are still being unraveled. To elucidate these relationships, we studied 16 human genes containing myogenic DMRs by analyzing profiles of their epigenetics and transcription and quantitatively assaying 5-hydroxymethylcytosine (5 hmC) and 5-methylcytosine (5 mC) at specific sites in these genes in skeletal muscle (SkM), myoblasts, heart, brain, and diverse other samples...
December 2, 2016: Epigenetics: Official Journal of the DNA Methylation Society
Margit Fuchs, Marie-Chloé Boulanger, Herman Lambert, Jacques Landry, Josée N Lavoie
Cellular processes such as mitosis and cell differentiation are governed by changes in cell shape that largely rely on proper remodeling of the cell cytoskeletal structures. This involves the assembly-disassembly of higher-order macromolecular structures at a given time and location, a process that is particularly sensitive to perturbations caused by overexpression of proteins. Methods that can preserve protein homeostasis and maintain near-to-normal cellular morphology are highly desirable to determine the functional contribution of a protein of interest in a wide range of cellular processes...
2016: Journal of Visualized Experiments: JoVE
Wenqiang Sun, Huilan Huang, Shengchao Ma, Xiang Gan, Mou Zhu, Hehe Liu, Liang Li, Jiwen Wang
The Akirin gene family normally contains two members that are essential to myoblast differentiation. Noticeably, the avian Akirin gene family comprises only one gene (Akirin2), However, it remains unknown whether avian Akirin gene family still has the function of Akirin1; moreover, it is still unclear whether and how Akirin2 plays a role in myoblast proliferation and differentiation. Interestingly, the unexpected functions of duck Akirin2 were revealed in the present study. The Real-time PCR results showed that between 12 and 48h during the process of duck myoblasts differentiation, the overexpression of Akirin2 did not significantly increase the expression of myogenic regulatory factors...
October 2016: International Journal of Biochemistry & Cell Biology
Petr Dmitriev, Ekaterina Kiseleva, Olga Kharchenko, Evgeny Ivashkin, Andrei Pichugin, Philippe Dessen, Thomas Robert, Frédérique Coppée, Alexandra Belayew, Gilles Carnac, Dalila Laoudj-Chenivesse, Marc Lipinski, Andrei Vasiliev, Yegor S Vassetzky
We performed transcriptome profiling of human immortalized myoblasts (MB) transiently expressing double homeobox transcription factor 4 (DUX4) and double homeobox transcription factor 4 centromeric (DUX4c) and identified 114 and 70 genes differentially expressed in DUX4- and DUX4c-transfected myoblasts, respectively. A significant number of differentially expressed genes were involved in inflammation, cellular migration and chemotaxis suggesting a role for DUX4 and DUX4c in these processes. DUX4 but not DUX4c overexpression resulted in upregulation of the CXCR4 (C-X-C motif Receptor 4) and CXCL12 (C-X-C motif ligand 12 also known as SDF1) expression in human immortalized myoblasts...
October 4, 2016: Oncotarget
Yung Hyun Choi
Preclinical Research The aim of the present study was to evaluate the effects of berberine hydrochloride (BBH), an isoquinoline alkaloid that can be isolated from a variety of herbs, on hydrogen peroxide (H2 O2 )-induced oxidative stress in C2C12 myoblasts and to investigate the molecular mechanisms involved in this process, especially the expression of the Nrf2/HO-1 pathway. BBH preconditioning attenuated H2 O2 -induced growth inhibition and DNA damage as well as apoptosis in C2C12 cells via suppression of the accumulation of intracellular reactive oxygen species (ROS)...
September 2016: Drug Development Research
Petr Dmitriev, Yara Bou Saada, Carla Dib, Eugénie Ansseau, Ana Barat, Aline Hamade, Philippe Dessen, Thomas Robert, Vladimir Lazar, Ruy A N Louzada, Corinne Dupuy, Vlada Zakharova, Gilles Carnac, Marc Lipinski, Yegor S Vassetzky
Facioscapulohumeral dystrophy (FSHD) is one of the three most common muscular dystrophies in the Western world, however, its etiology remains only partially understood. Here, we provide evidence of constitutive DNA damage in in vitro cultured myoblasts isolated from FSHD patients and demonstrate oxidative DNA damage implication in the differentiation of these cells into phenotypically-aberrant myotubes. Double homeobox 4 (DUX4), the major actor in FSHD pathology induced DNA damage accumulation when overexpressed in normal human myoblasts, and RNAi-mediated DUX4 inhibition reduced the level of DNA damage in FSHD myoblasts...
August 9, 2016: Free Radical Biology & Medicine
Rui Liang, Wei Dong, Xiaopeng Shen, Xiaoping Peng, Angie G Aceves, Yu Liu
Myotonic dystrophy 1 (DM1) is a common form of muscular dystrophy. Although several animal models have been established for DM1, myoblast cell models are still important because they offer an efficient cellular alternative for studying cellular and molecular events. Though C2C12 myoblast cells have been widely used to study myogenesis, resistance to gene transfection, or viral transduction, hinders research in C2C12 cells. Here, we describe an optimized protocol that includes daily maintenance, transfection and transduction procedures to introduce genes into C2C12 myoblasts and the induction of myocyte differentiation...
2016: Journal of Visualized Experiments: JoVE
Ge Gao, Christopher D Moyes
Cold acclimation in fish typically increases muscle mitochondrial enzymes. In mammals, stressors that increase mitochondrial content are mediated though transcriptional regulators, including nuclear respiratory factor-1 (NRF-1). Focusing on the goldfish gene for cytochrome c oxidase (COX) subunit 4-1, we analysed the regulatory regions in various contexts to identify a mechanistic link between NRF-1 and cold-induced mitochondrial proliferation. Promoter analysis implicated two putative NRF-1 sites: one in the proximal promoter and a second in exon 1, which encodes the 5' untranslated region (5'-UTR)...
October 1, 2016: Journal of Experimental Biology
Ruifeng Fan, Haidong Yao, Xia Zhao, Changyu Cao, Tianshu Yang, Yilin Luan, Ziwei Zhang, Shiwen Xu
The aim of the present study was to clarify the effect of Selenoprotein K (Selk) silencing on the mRNA expression of 25 selenoproteins in chicken myoblasts. The specific small interfering RNA (siRNA) for Selk gene was designed and transfected into chicken myoblasts. Post-transfection mRNA expression of 25 selenoproteins was determined at various time periods i.e., 24, 48 and 72 h. Moreover, based on the results of expression of 25 selenoproteins, correlation analysis and principal component analysis (PCA) were used for further analysis...
August 2016: Biometals: An International Journal on the Role of Metal Ions in Biology, Biochemistry, and Medicine
G N Sui, Z W Liu, T Wang, X Li, X B Ding, X F Liu, H Guo
The CMYA1 (cardiomyopathy-associated protein 1) is an actin-binding protein that plays a vital role in cardiac morphogenesis. CMYA1 is expressed specifically in the myocardial and skeletal muscle and is up-regulated in injured muscle. We therefore speculated that the bovine CMYA1 promoter might be muscle-specific. In this study, the promoter (+20/-1135) region of the bovine CMYA1 gene was cloned into a pEGFP-1 vector, and we found that the EGFP was observed only in C2C12 and myoblast cells. Thus, the CMYA1 promoter is muscle-specific...
June 3, 2016: Genetics and Molecular Research: GMR
Huijing Yang, Zhenhua Zhang, Liangyi Xue
Follistatin is a secreted glycoprotein, which involved in numerous physiological activities as an antagonist of the transforming growth factor-β superfamily. However, little is known about the regulation mechanism of follistatin in fish. In this study we cloned and analyzed part of the 5' flanking region of the follistatin gene in Larimichthys crocea. Sequence analysis revealed several putative binding sites for transcription factors including activator protein 1 (AP1), myogenic differentiation factor (MyoD), stimulating protein 1 (SP1), and sex determining gene on the mammalian Y chromosome (SRY) in the cloned fragment...
September 2016: DNA and Cell Biology
Yoo-Hwan Lee, Jung-Hee Kim, Choon-Ho Song, Kyung-Jeon Jang, Cheol-Hong Kim, Ji-Sook Kang, Yung-Hyun Choi, Hyun-Min Yoon
OBJECTIVES: The mushroom Ganoderma lucidum has been widely used as a traditional herbal medicine for many years. Although several studies have focused on the anti-oxidative activity of this mushroom, the molecular mechanisms underlying its activity have not yet been clearly established. The present study investigated the cytoprotective effect of ethanol extract of Ganoderma lucidum (EGL) against oxidative stress (hydrogen peroxide, H2O2) and elucidated the underlying mechanisms in a C2C12 myoblast cell line...
March 2016: Journal of Pharmacopuncture
Mulan Qahar, Yuko Takuma, Wataru Mizunoya, Ryuichi Tatsumi, Yoshihide Ikeuchi, Mako Nakamura
We previously showed that Semaphorin 3A (Sema3A) expression was induced when quiescent muscle satellite cells were stimulated by hepatocyte growth factor and became activated satellite cells (ASCs). However, how Sema3A regulates genes in the early phase of ASCs remains unclear. In this study, we investigated whether Sema3A signaling can regulate the early phase of ASCs, an important satellite cell stage for postnatal growth, repair, and maintenance of skeletal muscle. We showed that expression of the myogenic proliferation regulatory factors Pax7 and Myf5 was decreased in myoblasts transfected with Sema3A siRNA...
June 2016: FEBS Open Bio
Wei Chen, Houqiang Xu, Xiang Chen, Zhongwei Liu, Wen Zhang, Dan Xia
Uncoupling protein 3 (UCP3) is mainly expressed in muscle. It plays an important role in muscle, but less research on the regulation of cattle UCP3 has been performed. In order to elucidate whether cattle UCP3 can be regulated by muscle-related factors, deletion of cattle UCP3 promoter was amplified and cloned into pGL3-basic, pGL3-promoter and PEGFP-N3 vector, respectively, then transfected into C2C12 myoblasts cells and UCP3 promoter activity was measured using the dual-Luciferase reporter assay system. The results showed that there is some negative-regulatory element from -620 to -433 bp, and there is some positive-regulatory element between -433 and -385 bp...
May 5, 2016: International Journal of Molecular Sciences
Carlos J Rodriguez-Ortiz, Julio C Flores, Joanna A Valenzuela, Gema J Rodriguez, Joannee Zumkehr, Diana N Tran, Virginia E Kimonis, Masashi Kitazawa
Valosin-containing protein (VCP) mutations cause inclusion body myopathy with Paget disease and frontotemporal dementia. However, the mechanisms by which mutant VCP triggers degeneration remain unknown. Here, we investigated the role of VCP in cellular stress and found that the oxidative stressor arsenite and heat shock-activated stress responses evident by T-intracellular antigen-1-positive granules in C2C12 myoblasts. Granules also contained phosphorylated transactive response DNA-binding protein 43, ubiquitin, microtubule-associated protein 1A/1B light chains 3, and lysosome-associated membrane protein 2...
June 2016: American Journal of Pathology
Qiying Chen, Yue Zhou, A Mark Richards, Peipei Wang
Timely reperfusion in acute myocardial infarction has improved clinical outcomes but the benefits are partially offset by ischemia-reperfusion injury (I/R). MiRNA regulates mRNA of multiple effectors within injury and survival cell signaling pathways. We have previously reported the protective effects of miRNA-221 in I/R injury. The purpose of this study was to explore the mechanisms underlying cardioprotection of miR-221. Myoblast H9c2 and neonatal rat ventricular myocytes (NRVM) were subjected to 0.2% O2 hypoxia followed by 2 h of re-oxygenation (H/R)...
May 20, 2016: Biochemical and Biophysical Research Communications
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