C2C12 culture: myogenesis timecourse and shRp58 treatment
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE12993 | GEO | 2009/07/31
SECONDARY ACCESSION(S): PRJNA112605
REPOSITORIES: GEO
Similar Datasets
Project description:To predict Rp58-regulated gene involved in myogenesis, RNA profiling experiments were performed, comparing RNA derived from C2C12 with or without expressing shRNA for Rp58. As a result, 271 genes were upregulated in C2C12 stably expressing shRNA-Rp58 cells compared with control C2C12 cells. As Rp58 is repressor in C2C12, we hypothesized that Rp58 regulates gene cluster which expression is downregulated in accordance with Rp58 expression and myogenesis progression. In this regard, we also characterized dynamic gene expression patterns during myogenesis by microarray at 4 different stage (GM, day 0, 2, 4) of C2C12 myogenesis assays and found that 399 genes expression is characterized as downregulation pattern during myogenesis. Importantly, this down regulation gene set and upregulated genes by shRNA for Rp58 were highly overlapped. Experiment Overall Design: C2C12 murine skeletal muscle cells were purchased from American Type Experiment Overall Design: culture Collection (ATCC). These cells were mainteined in GM (DMEM Experiment Overall Design: supplemented with 10% FBS). Cells were grown in GM and after reaching Experiment Overall Design: counfluence, the medium was switched to DM (DMEM supplemented with 2% hourse serum) and further incubated. The medium was changed every 2 days. Culture was performed by using within five passages cells. For the experiment of shRNA for Rp58, transfection was performed by using Lipofectamin 2000 (Invitrogen). Stable transfectants were obtained by selection of the transfected C2C12 cells for two weeks. Experiment Overall Design: Microarray analysis - RNA was isolated as described from C2C12, and cRNA was synthesized. 10 ug of cRNA were hybridized to Affymetrix mouse 430 2.0 arrays. Intensity values were quantified using RMA algorithm. Experiment Overall Design: MAPPFinder (www.genmapp.org) was used to integrate expression data with known pathways.
2009-08-09 | E-GEOD-12993 | biostudies-arrayexpress
Project description:ADAMTSL2 mutations cause geleophysic dysplasia, which is characterized by short stature, pseudomuscular build, joint stiffness and tight skin. To elucidate the role of ADAMTSL2 in skeletal muscle myogenesis, we used C2C12 myoblasts, which differentiate and form myotubes when serum is reduced as a model system for myogenesis. Adamtsl2 was depleted by stably expressing shRNA targeting Adamtsl2 mRNA. Upon shRNA-mediated depletion of Adamtsl2, C2C12 myoblasts did not differentiate and did not form myosin heavy chain-positive myotubes in cell culture.
2021-10-16 | GSE185894 | GEO
Project description:Myogenesis is a tightly controlled process involving the transcriptional activation and repression of thousands of genes. Although many components of the transcriptional network are known for the later phases of myogenesis, relatively little work has described the transcriptional landscape within the first 24 hours, when myoblasts commit to differentiate. Through dense temporal sampling of differentiating C2C12 myoblasts, we identify 266 transcriptional regulators (TRs) whose expression is altered within the first 12 hours of myogenesis. A high-content shRNA screen of 76 TRs involving 427 stable lines identified 48 genes whose knockdown significantly inhibits differentiation of C2C12 myoblasts. These include known regulators of myogenesis (Myod1, Myog and Myf5), as well as 26 regulators not previously associated with the process. Of the TRs differentially expressed within the first 24 hours, two-thirds inhibited differentiation when knocked down. Surprisingly, a similar proportion (67%) of shRNAs targeting TRs whose expression did not change during differentiation also inhibited myogenesis, suggesting that both stably and differentially expressed TRs are essential for this complex differentiation program. This implies that microarray-based approaches that concentrate functional validation studies on differentially-expressed genes will fail to identify many genes that are critically implicated in complex biological processes. C2C12 myoblasts were differentiated into myotubes and sampled at various timepoints for gene expression measurement on Illumina Mouse WG-6v1.1 arrays. Cells grown in separate plates were harvested at 12 different timepoints: t_0h, t_1h, t_1.5h, t_2h, t_3h, t_6h, t_9h, t_12h, t_24h, t_48h, t_96h, t_144h. All harvests were performed in triplicate using growths from successive passages.
2012-03-21 | E-GEOD-17038 | biostudies-arrayexpress
Project description:Analysis of Early Myogenesis Reveals an Extensive Set of Transcriptional Regulators Whose Knock-down Can Inhibit Differentiation Myogenesis is a tightly controlled process involving the transcriptional activation and repression of thousands of genes. Although many components of the transcriptional network are known for the later phases of myogenesis, relatively little work has described the transcriptional landscape within the first 24 hours, when myoblasts commit to differentiate. Through dense temporal sampling of differentiating C2C12 myoblasts, we identify 266 transcriptional regulators (TRs) whose expression is altered within the first 12 hours of myogenesis. A high-content shRNA screen of 76 TRs involving 427 stable lines identified 48 genes whose knockdown significantly inhibits differentiation of C2C12 myoblasts. These include known regulators of myogenesis (Myod1, Myog and Myf5), as well as 26 regulators not previously associated with the process. Of the TRs differentially expressed within the first 24 hours, two-thirds inhibited differentiation when knocked down. Surprisingly, a similar proportion (67%) of shRNAs targeting TRs whose expression did not change during differentiation also inhibited myogenesis, suggesting that both stably and differentially expressed TRs are essential for this complex differentiation program. This implies that microarray-based approaches that concentrate functional validation studies on differentially-expressed genes will fail to identify many genes that are critically implicated in complex biological processes. C2C12 myoblasts were differentiated into myotubes and sampled at various time points for gene expression measurement on MOE-430v2 chips. Cells grown in separate plates were harvested at 14 different time points: t_-24h, t_0h, t_0.5h, t_1h, t_1.5h, t_2h, t_3h, t_6h, t_9h, t_12h, t_24h, t_48h, t_96h, t_144h. Cells were also pre-treated with 50uM cycloheximide 1 hour prior to inducing differentiation and harvested at two time points: t_chx_1h, t_chx_3h. All harvests were performed in triplicate using growths from successive passages.
2012-03-21 | E-GEOD-16992 | biostudies-arrayexpress
Project description:Myogenesis is a tightly controlled process involving the transcriptional activation and repression of thousands of genes. Although many components of the transcriptional network are known for the later phases of myogenesis, relatively little work has described the transcriptional landscape within the first 24 hours, when myoblasts commit to differentiate. Through dense temporal sampling of differentiating C2C12 myoblasts, we identify 266 transcriptional regulators (TRs) whose expression is altered within the first 12 hours of myogenesis. A high-content shRNA screen of 76 TRs involving 427 stable lines identified 48 genes whose knockdown significantly inhibits differentiation of C2C12 myoblasts. These include known regulators of myogenesis (Myod1, Myog and Myf5), as well as 26 regulators not previously associated with the process. Of the TRs differentially expressed within the first 24 hours, two-thirds inhibited differentiation when knocked down. Surprisingly, a similar proportion (67%) of shRNAs targeting TRs whose expression did not change during differentiation also inhibited myogenesis, suggesting that both stably and differentially expressed TRs are essential for this complex differentiation program. This implies that microarray-based approaches that concentrate functional validation studies on differentially-expressed genes will fail to identify many genes that are critically implicated in complex biological processes.
2012-03-21 | GSE17038 | GEO
Project description:Analysis of Early Myogenesis Reveals an Extensive Set of Transcriptional Regulators Whose Knock-down Can Inhibit Differentiation Myogenesis is a tightly controlled process involving the transcriptional activation and repression of thousands of genes. Although many components of the transcriptional network are known for the later phases of myogenesis, relatively little work has described the transcriptional landscape within the first 24 hours, when myoblasts commit to differentiate. Through dense temporal sampling of differentiating C2C12 myoblasts, we identify 266 transcriptional regulators (TRs) whose expression is altered within the first 12 hours of myogenesis. A high-content shRNA screen of 76 TRs involving 427 stable lines identified 48 genes whose knockdown significantly inhibits differentiation of C2C12 myoblasts. These include known regulators of myogenesis (Myod1, Myog and Myf5), as well as 26 regulators not previously associated with the process. Of the TRs differentially expressed within the first 24 hours, two-thirds inhibited differentiation when knocked down. Surprisingly, a similar proportion (67%) of shRNAs targeting TRs whose expression did not change during differentiation also inhibited myogenesis, suggesting that both stably and differentially expressed TRs are essential for this complex differentiation program. This implies that microarray-based approaches that concentrate functional validation studies on differentially-expressed genes will fail to identify many genes that are critically implicated in complex biological processes.
2012-03-21 | GSE16992 | GEO
Project description:To predict RP58-regulated genes involved in skeletal myogenesis, RNA profiling experiments were performed, comparing RNA derived from skeletal muscle tissue of a RP58+/+ mouse to that from a RP58 knockout (KO) mouse at E18.5. Importantly, well-known dominant-negative inhibitors of muscle differentiation, the Id family of genes (Id1/Id2/Id3), were upregulated in the RP58 KO muscle. On the contrary, a number of muscle differentiation-related genes, such as Ckm, troponin and Myosin, were downregulated in the same sample. These results indicate that the repressor protein RP58 is important for muscle terminal differentiation, possibly suppressing the gene expression of muscle differentiation genes such as the Ids. Keywords: Knockout tissue
2009-07-31 | GSE16595 | GEO
Project description:This study profiled the expression of miRNA during myogenesis of the C2C12 mouse myoblast cell line. Cells were harvest at different stages during myogenesis including proliferating, confluence, 1 day post-differentiation induction, 2 day post-differentiation induction and 4 days post-differentiation induction. Keywords: time course This experiment was a reference design miRNA microarray where all time points were compared to a pooled proliferating C2C12 sample, made up of 4 proliferating biological replicates. Each time point had four bioloigcal replicates of which two were labelled with Cy3 and two were labelled with Cy5.
2010-05-26 | E-GEOD-9449 | biostudies-arrayexpress
Project description:This study profiled the expression of miRNA during myogenesis of the C2C12 mouse myoblast cell line. Cells were harvest at different stages during myogenesis including proliferating, confluence, 1 day post-differentiation induction, 2 day post-differentiation induction and 4 days post-differentiation induction. Keywords: time course
2008-06-30 | GSE9449 | GEO
Project description:Myogenesis is governed by signalling networks whose regulations are tightly controlled in a time-dependent manner. While different protein kinases have been identified to regulate various aspects of myogenesis, knowledge on the global signalling networks and their downstream substrates during myogenesis remains incomplete. Here, we map the myogenic differentiation of C2C12 cells using mass spectrometry (MS)-based phosphoproteomics and proteomics. From these data, we infer global kinase activity and predict substrates of key kinases that are involved in myogenesis. We found that multiple mitogen-activated protein kinases (MAPKs) mark the initial wave of signalling cascades. Further phosphoproteomic and proteomic profiling with MAPK1/3 and MAPK8/9 specific inhibitions unveil their shared and distinctive roles on myogenesis.
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