For each cell, volume http://www.selleckchem.com/products/ABT-888.html and flattening were measured. The relative distance of the centromere hybridisation signal was calculated from the nuclear centre, based on the division of the nuclear diameter on equal sections (Figure 1). Volume and flattening samples for both, myoblasts and myotubes, did not undergo a normal distribution (Shopiro-Wilk test for normality, p-value<0.001 for all four samples), therefore, we decided to use a non-parametrical analysis. Statistical significance of a morphological analysis of the nuclei volume and the flattening was calculated by Mann-Whitney U test. The nucleus of each cell was divided into five co-centric shells of the same volume. Then, centromeres signals were assigned to appropriate shells, creating a signal distribution in cell nuclei.

The centromere signal distribution was then analyzed with the chi-square test. Figure 1 Scheme of 3D analysis of interphase nuclei. Microarray Assay RNA was obtained from in vitro cultured myoblasts and myocytes using the TriReagent (Sigma-Aldrich, St. Louis, USA) according to the manufacturer��s protocol. Evaluation of RNA integrity and quantity was conducted using the BioAnalyser (Agilent, Santa Clara, USA) and the NanoDrop spectrophotometer (Nanodrop, Wilmington, USA). The samples were prepared in triplicate. The single stranded DNA was generated using the Ambion WT Expression Kit (Ambion, Austin, USA), followed by fragmentation and labelling of cDNA using the Affymetrix GeneChip WT Terminal Labelling Kit (Affymetrix, Santa Clara, CA).

Array hybridisation, washing and scanning were conducted according to manufacturer��s protocol using high-density arrays GeneChip Human Gene 1.0 ST (Affymetrix, Santa Clara, CA). After washing, microarrays were scanned using the GeneChip Scanner 3000 (Affymetrix, Santa Clara, CA). The data shown in this publication have been deposited in NCBI��s Gene Expression Omnibus and are accessible through GEO Series accession number “type”:”entrez-geo”,”attrs”:”text”:”GSE45819″,”term_id”:”45819″GSE45819 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=”type”:”entrez-geo”,”attrs”:”text”:”GSE45819″,”term_id”:”45819″GSE45819). Data Analysis The CEL files obtained were analysed with the Expression Console software (Affymetrix, Santa Clara, CA) by using Robust Multi-chip Analysis (RMA) to correct the background.

Quantile normalisation was performed and the results obtained were normalised while the adjusted set of data was expressed on a logarithmic scale. For subsequent analysis, Subio software (Subio, Japan) was used. GSK-3 Differential expression analysis was conducted for transcripts showing at least a 2-fold difference between groups and statistical significance was determined by the Benjamini-Hochberg algorithm for multiple testing to adjust the p-value defined by the t-test (p<0.01).