ABSTRACT: Gene expression profiling in differentiating C2C12 cells comparing control cells and MUNC-deprived cells RNA samples were isolated from 4 C2C12 samples: proliferating control (siC) cells, differentiating siC cells, proliferating and differentiating MUNC depleted (siMUNC) cells
Project description:We newly identified skeletal muscle differentiation-associated miRNAs by comparing miRNA expression profile between C2C12 cell and Wnt4-overexpressing C2C12 cell. miR-487b, miR-3963 and miR-6412 are significantly down-regulated in differentiating C2C12 cells, and transfection of their mimics resulted in reduced expression of myogenic differentiation markers including Troponin T, myosin heavy chain fast and slow type. Single analysis for each condition (proliferating C2C12 cells, differentiating C2C12 cells, proliferating Wnt4-overexpressing C2C12 subline cells
Project description:Wnt/beta-catenin signaling is involved in various aspects of skeletal muscle development and regeneration. Using C2C12 cells, we examined intracellular signaling and gene transcription during myoblast proliferation and differentiation. The results of the present studies suggest that Wnt signaling is interacting with TGF-beta superfamily signaling through Smad activation. Single analysis for each condition (proliferating C2C12 cells, differentiating C2C12 cells, proliferating Wnt4-overexpressing C2C12 subline cells).
Project description:We used microarrays to characterize the global changes in gene expression in C2C12 cells due to siRNA knockdown of long non-coding RNA H19 Control siRNA or siRNA specific for mouse H19 were transfected into day1 differentiating C2C12 myoblasts in triplicates. 40 H later total RNAs were isolated and subjected with microarray analysis.
Project description:Acetoacetate (AA) is a ketone body and acts as a fuel to supply energy for cellular activity of various tissues. Here, we performed differential RNA-Seq analyses of proliferating and differentiating C2C12 myoblasts in the present or absent of AA. Differentially regulated genes and gene set enrichment analysis revealed that altered expression of the AA-mediated genes in both GM (growth medium) and DM (differentiation medium) were highly enriched in the signaling pathways associated with cell proliferation and differentiation, however, the genes encoding enzymes in biochemical pathways of ketolysis for energy production were not transcriptionally changed in the cells cultured in either GM or DM in response to AA. Notably the genes differentially expressed between GM and DM were also significantly regulated by AA. This indicates that AA-mediated myogenic cell function by transcriptional regulation of the genes required for cell proliferation and differentiation. Taken together, the data from our multi-parameter analyses consistently support the notion that AA plays a non-metabolic role in regulating muscle cell function. We sequenced mRNA from C2C12 cells in GM or DM treated with AA for 12 h or not to generate genes response to AA during C2C12 proliferation and differentiation. Three of duplication was performed. Examination of mRNA levels in proliferating or differentiating C2C12 cells between AA-treated groups and control groups.
Project description:Transcriptional profiling of mouse postnatal SVZ NSCs comparing WT NSCs with KO NSCs under proliferating/undifferentiated states as well as differentiating conditions. Goal was to determine Dnmt3a-dependent gene expression changes in postnatal SVZ NSCs Two-condition experiment with a dye-swap design, WT NSCs vs. KO NSCs. Biological replicates: 4 replicates under proliferating/undifferentiation conditions, 2 replicates under differentiating conditions.
Project description:Human epidermal keratinocytes undergo tightly controlled program of cell differentiation, leading to the formation of cornified envelope. Primary keratinocytes in vitro, under calcium stimulation mimic the differentiation program observed in vivo. Analysis of the transcription profile of two cell population, such as proliferating cells and differentiating cells helps to discover new genes implicated in that process and to understand the mechanisms of regulation of the keratinocyte differentiation. Primary human keratinocytes were cultured under proliferating (Day 0, sub-confluent cells) and differentiating (seven days of high calcium medium) conditions. As a source of cells, we used normal skin from different body sites: back, foreskin, palmoplantar. RNA extracted from cultured primary human keratinocytes were isolated from five different donors. We compared the expression profiles of proliferating versus differentiating keratinocytes.
Project description:Steady-state RNA levels are a result of RNA synthesis and degradation. The importance of transcription-factor mediated induction or repression of mRNA synthesis is well established, but the role and mechanisms of RNA degradation are less well understood. We globally evaluated the RNA decay rates in proliferating and differentiated mouse myoblasts on whole-genome Affymetrix exon arrays, allowing for the assessment of directionality of RNA degradation and the detection of splice variant-specific differences in RNA decay rates. We found large differences in decay rates. mRNAs coding for proteins involved in signal transduction and transcriptional regulation have shortest half lives, whereas mRNAs coding for DNA replication enzymes and muscle contraction proteins are among the most stable. Many genes differentially expressed between proliferating and differentiated myoblasts demonstrate major differences in RNA decay rates. Quantitative PCR experiments confirmed the higher stability of transcripts with increased expression levels. RNA degradation has no apparent preferential directionality. RNA degradation appears to affect the ratio of different splice variants. For example, Itga7 isoforms with higher abundance in differentiated than in proliferating cells are more stable in differentiated cells, despite the sharing of a common 3’ untranslated region. Thus, where it was previously thought that the abundance of different splice isoforms was mainly controlled by tissue-specific splicing factors, we now demonstrate that isoforms may be produced at comparable levels but degraded with different efficiencies, depending on the differentiation status of the cells. Our results indicate that control of RNA degradation rates contributes significantly to the differentiation stage-dependent differences in abundance of transcripts and splice variants. Keywords: total RNA expression profiling; cultured cells We analyzed proliferating C2C12 cells and C2C12 cells differentiated into myotubes by serum starvation (8 days after induction of differentiation). We analyzed 7 time points after addition of actionmycin D (0, 10, 20, 30, 60, 150, 480 minutes). There were two biological replicates per condition.
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.
Project description:Selective genetic ablation of the SIRT1 deacetylase domain in skeletal muscle results in increased H4K16 acetylation and deregulated activation of the myogenic program in satellite cells To establish the role of the deacetylase SIRT1 in skeletal muscle we examined the genome wide distribution of H4K16ac in quiescent (FI) and proliferating (Cul) satellite cells isolated from WT mice (C57Bl/6 background) and SIRT1mKO (generated via breeding of Pax7cre/+ knock-in mice with mice containing the floxed exon 4 SIRT1 allele). We also analyzed the distribution of SIRT1 in quiescent and proliferating FACS isolated WT satellite cells (two replicates). We generated the mRNA profiles (at least two replicate for each experiment) of FACS isolated quiescent, proliferating and differentiating (1 day in differentiation medium) satellite cells of WT mice and SIRT1mKO. The selective genetic ablation of the SIRT1 deacetylase domain in skeletal muscle results in increased H4K16 acetylation and deregulated activation of the myogenic program.
Project description:CUGBP1 and MBNL1 are developmentally regulated RNA-binding proteins that are causally associated with myotonic dystrophy type 1. Using HITS-CLIP anlysis, we found CUGBP1 and MBNL1 preferentially bind to alternatively spliced introns and exons, as well as to the 3' UTRs. To analyze more directly the role of CUGBP1/MBNL1 binding in alternative splicing, we performed exon array analysis in C2C12 cells using expression arrays. We analyzed total RNA of C2C12 cells treated with control-, Cugbp1- or Mbnl1-siRNA. RNA was harvested 48 hrs after transfection.