Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

MicroRNA profiling by array of mouse skeletal muscle cells treated with TNF-alpha or IGF-I

ABSTRACT: Murine skeletal muscle cells pmi28 were cultured in growth medium (HamM-^Rs F10, supplemented with 20 % FCS, 2 mM L-glutamine, and 1 % Penicillin / Streptomycin). Myoblasts were cultured on laminin-1 coated dishes for an additional 24 h before switching a fraction of dishes to differentiation medium (DMEM medium containing 2 % horse serum, 2 mM L glutamine, and 1 % Penicillin / Streptomycin) and to differentiation medium containing either 2 x 103 U/ml mouse recombinant TNF-? (Roche Applied Science), or 5 M-5g/ml mouse recombinant IGF-I (Sigma-Aldrich), or carrier only. pmi28 cells (TNF-? and IGF-I treatments as well as controls) were harvested 24 h after the induction of differentiation. The experiments were performed in quadruplicates or triplicates, respectively.

ORGANISM(S): Mus musculus

SUBMITTER: Swanhild Meyer

PROVIDER: E-MTAB-1114 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Publications

Profound effect of profiling platform and normalization strategy on detection of differentially expressed microRNAs--a comparative study.

Meyer Swanhild U SU Kaiser Sebastian S Wagner Carola C Thirion Christian C Pfaffl Michael W MW

PloS one 20120618 6

<h4>Background</h4>Adequate normalization minimizes the effects of systematic technical variations and is a prerequisite for getting meaningful biological changes. However, there is inconsistency about miRNA normalization performances and recommendations. Thus, we investigated the impact of seven different normalization methods (reference gene index, global geometric mean, quantile, invariant selection, loess, loessM, and generalized procrustes analysis) on intra- and inter-platform performance ...[more]

PMID: 22723911

Similar Datasets

Project description:Treatment with insulin-like growth factor-I (IGF-I) during feed deprivation attenuates the weight loss response in both mammals and fish, therefore the reduction in plasma IGF-I concentrations during fasting likely serves as a signal that contributes to the catabolic response. To better understand the physiological mechanisms responsible for this effect rainbow trout were administered IGF-I during a 2 wk period of feed deprivation and changes in gene expression in white muscle were determined using microarray analysis. Weight loss was reduced by 15% (P<0.05) in IGF-I treated fish. A total of 440 transcripts were identified as differentially regulated (P<0.05) between saline and IGF-I treated fish. Genes related to protein degradation were down-regulated and included protease and peptidase genes and genes involved in ubiquitin-proteasome and cathepsin-mediated proteolytic pathways. IGF-I increased expression of myosin binding protein H and coronin-1C, while decreasing expression of other myofibrillar and cytoskeleton-associated genes like troponin-C and parvalbumin-2. Polyadenylate-binding protein 2, a transcription factor that positively regulates myoD and myogenin expression, was upregulated with IGF-I treatment. Additional genes that were differentially regulated are associated with lipid and carbohydrate metabolism, mitochondrial biogenesis and electron transport, gene transcription, signal transduction and regulation of apoptosis. In summary, these data suggest that IGF-I plays a central role in regulating protein degradation, especially via the ubiquitin-proteasomal pathway, and that reductions in protein degradation and the subsequent effects on other physiological systems are largely responsible for the IGF-I-induced reduction in weight loss. Eight approximately 1-year old full-sibling rainbow trout families were housed in individual tanks, according to family, at the National Center for Cool and Cold Water. Each fish was pit-tagged at 7-months of age. At the beginning of the study, fish were anesthetized with tricaine methanesulfonate (MS-222), weighed, and an osmotic pump was surgically inserted into the peritoneal cavity of each fish (n=4 fish/family/treatment, N=16 fish). The osmotic pump contained either recombinant human IGF-I, which released hormone at 25 ug/kg/day, or saline, which was the vehicle used to resuspend IGF-I. Feed was withheld 2-days prior to surgery and for the experimental 2 week period. Fish were harvested using as overdose of MS-222, weighed, and white muscle samples were removed, frozen in liquid N, and stored at -80 C until analysis. Muscle RNA was isolated from muscle of the two families that exhibited the greatest difference in weight loss between IGF-I and saline treatments; families 64 and 107. Hybridizations were performed using a dye-swap design with an IGF-I and saline treated fish within the same family, therefore a total of 16 slides were hybridized using Alexa 647 and Alexa 555 dyes.

Project description:Ewing Sarcoma is caused by a pathognomonic genomic translocation that places an N-terminal EWSR1 gene in approximation with one of several ETS genes (typically FLI1). This aberration, in turn, alters the transcriptional regulation of more than five hundred genes and perturbs a number of critical pathways that promote oncogenesis, cell growth, invasion, and metastasis. Among them, translocation-mediated up-regulation of the insulin-like growth factor receptor 1 (IGF-1R) and mammalian target of rapamycin (mTOR) are of particular importance since they work in concert to facilitate IGF-1R expression and ligand-induced activation, respectively, of proven importance in ES transformation. When used as a single agent in Ewing sarcoma therapy, IGF-1R or mTOR inhibition leads to rapid counter-regulatory effects that blunt the intended therapeutic purpose. Therefore, identify new mechanisms of resistance that are used by Ewing sarcoma to evade cell death to single-agent IGF-1R inhibition might suggest a number of therapeutic combinations that could improve its clinical activity. TC32 and TC71 ES clones with acquired resistance to OSI-906 or NVP-ADW-742 were generated by maintaining the corresponding parental cell lines with increasing concentrations of the agents (up to 2.3 Î¼M for OSI-906, 1.5 Î¼M for NVP-ADW-742) for 7 months. All parental and acquired drug resistant cell lines were tested twice per year for mycoplasma contamination using the MycoAlert Detection Kit (Lonza Group Ltd.) according to the manufacturerâs protocol and validated using short-tandem repeat fingerprinting with an AmpFLSTR Identifier kit as previously described. Herein, we determine subtle differences in acquired mechanism of resistance by two promising small molecule inhibitors of IGF-1R/IR-Î±. OSI-906, which inhibits IGF-1R and IR, and NVP-ADW-742, which inhibits only IGF-1R, were evaluated using in vitro assays to decipher the mechanism(s) by which IGF-1R inhibition induces drug resistance in Ewing sarcoma cells. The preparation of extracted proteins from sensitive and acquired resistant Ewing sarcoma cells to OSI-906 and NVP-ADW-742 for reverse-phase protein lysate array (RPPA) analysis were prepared using the same array. Lysates were processed, spotted onto nitrocellulose-coated FAST slides, probed with 115 validated primary antibodies, and detected using a DakoCytomation-catalyzed system with secondary antibodies. MicroVigene software program (VigeneTech) was used for automated spot identification, background correction, and individual spot-intensity determination. Expression data was normalized for possible unequal protein loading, taking into account the signal intensity for each sample for all antibodies tested. Log2 values were media-centered by protein to account for variability in signal intensity by time and were calculated using the formula log2 signal â log2 median. Principal component analysis was used to check for a batch effect and feature-by-feature two-sample t-tests were used to assess differences between sensitive and resistant cell lines to drug treatments. We also used feature-by-feature one-way analysis of variance (ANOVA) followed by the Tukey test to perform pair comparisons for all groups. Beta-uniform mixture models were used to fit the resulting p value distributions to adjust for multiple comparisons. The cutoff p values and number of significant proteins were computed for several different false discovery rates (FDRs). Biostatistical analyses comparing two groups were performed using an unpaired t-test with Gaussian distribution followed by the Welch correction. To distinguish between treatment groups, we used one-way ANOVA with the Geisser-Greenhouse correction. Differences with p values <0.05 were considered significant. Within clustered image maps (CIM), unsupervised double hierarchical clustering used the Pearson correlation distance and Wardâs linkage method as the clustering algorithm to link entities (proteins) and samples.

Dataset Information

MicroRNA profiling by array of mouse skeletal muscle cells treated with TNF-alpha or IGF-I

Publications

Profound effect of profiling platform and normalization strategy on detection of differentially expressed microRNAs--a comparative study.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure