Project description:In this study, the C2C12 cell line, a model used to study myogenesis and regeneration, was allowed to differentiate from myoblast precursor cells to myotubes. Cells were harvested at 3 different timepoints to perform ChIP-on-Chip of Six1, which is a key muscle regulator. We identified global loci bound by Six1 during skeletal myoblast differentiation. C2C12 Myoblasts were allowed to differentiate into myotubes. Cells at three timepoints were harvested for ChIP-on-Chip, including myoblasts stage, 24h after differentiation and myotubes (96h after differentiation). Myotubes were detached from the undifferentiated myoblast reserve cells using diluted trypsin. 3 independent biological replicates were used for each time point experiment. A microarray set counts 3 arrays (Custom Arrays A, B and C) for a total of approximately 2.9 million probes.
Project description:In urodele amphibians, limb regeneration involves the dedifferentiation of muscle myotubes into single cells that may acquire pluripotent potential. We have employed small molecules (myoseverin and BIO) to attempt to reproduce this behavior in mammalian muscle culture. C2C12 myotubes derived from the C2C12 myoblast cell line were induced to undergo cellularization by myoseverin treatment, which destabilizes tubulin filaments. The GSK-3 inhibitor, BIO, was then used to induce dedifferentiation. Induce neuron formation; the cells were incubated with 250 nM reversine for 48 h, and neural induction media (DMEM/F12 supplemented with N2 (Invitrogen)) and 1.5 uM all-trans retinoic acid for 7 days. C2C12 murine myoblast cell line and 48 h 10 uM BIO treated C2C12 cellulate (derived by 20 M myoseverin treatment for 48 h) C2C12 myoblasts were differentiated into myotubes with 2%horse serum in DMEM for 8 days (from 2-4 d, 10 uM AraC treatment was also used to kill any remaining myoblasts). Next, myotubes were cellularized by 20 uM myoseverin treatment for 48 h. 24 h after myoseverin treatment, myotubes were treated with 10 uM BIO for 2d.
Project description:In urodele amphibians, limb regeneration involves the dedifferentiation of muscle myotubes into single cells that may acquire pluripotent potential. We have employed small molecules (myoseverin and BIO) to attempt to reproduce this behavior in mammalian muscle culture. C2C12 myotubes derived from the C2C12 myoblast cell line were induced to undergo cellularization by myoseverin treatment, which destabilizes tubulin filaments. The GSK-3 inhibitor, BIO, was then used to induce dedifferentiation. Induce neuron formation; the cells were incubated with 250 nM reversine for 48 h, and neural induction media (DMEM/F12 supplemented with N2 (Invitrogen)) and 1.5 uM all-trans retinoic acid for 7 days. C2C12 murine myoblast cell line and 48 h 10 uM BIO treated C2C12 cellulate (derived by 20 M myoseverin treatment for 48 h)
Project description:We show the application of 5mC antibody-based methylated DNA immunoprecipitation followed sequencing technology for high-through profiling of DNA methylation in mouse C2C12 myoblasts and myotubes. By analyzing the methylation status of immunoprecipitated DNA fragments, we generated genome-wide DNA methyaltion maps in mouse C2C12 myoblasts and myotubes. We find that DNA methylation levels in myoblasts at rDNA promter and coding regions are higher than that in myotubes but not changed in intergenic regions.