Clearly, changes in the nuclear localisation of the centromeres EPZ-5676 on five chromosomes (1, 3, 12, 17, X) were observed (p<0.05), whereas the centromeres of chromosomes 7 and 11 seemed to remain stable (>0.05). We could distinguish three schemes of centromere re-localisation; the first of these applied to the centromeres of chromosomes 1, 3 and 17, with a well�Cdefined movement towards the nuclear border, whereas the centromeres of chromosomes 12 and X were preferentially found in the intermediate shells of the nuclei, which defined the second type of re-localisation. The last group consisted of the centromeres of chromosomes 7 and 11, which were rather stable in their nuclear position. The apparent changes in the location of the centromere of chromosome 1 were noticed in the first shell, in which 19.

77% of the signal in myoblasts was observed, whereas only 4.55% of the signal was observed in myocytes. The same phenomena were observed in the case of chromosome 3; almost 26% of the centromeres were located in the inner shell in the undifferentiated cell state. However, after myocyte formation, only 7.89% of the centromeres were found in the centre of the nuclei. The arrangement of the centromeres of chromosomes 7 and 11 did not change during myogenesis. The outermost shell seemed to be rather unoccupied (from 1.89% to 9.82% centromeres of the investigated chromosomes), with the exception of the chromosome X centromere, in which 14.58% (myoblasts) and 19.15% (myocytes) of the centromeric signals were detected.

Interestingly, 10% of the centromeres of chromosome 12 were found in the outer shell of myocyte nuclei, but no centromeres were localized in myoblasts. The centromere of chromosome X also seemed to have moved towards the nuclear lamina during myogenesis; 23.96% of the centromere signals were located in the inner shell during myogenesis and only 7.45% were found in the inner shell after differentiation. The data have been summarised in Table 1 and illustrated in Figure 6. Figure 5 Cytogenetic maps of chosen chromosomes along with genes (loci) of interest marked. Figure 6 Comparison of the changes in chromosome centromere distribution, before (myoblasts Mb24 hrs ? red spots) and after (myocytes Mc7d ? yellow spots) cell differentiation. Table 1 Comparison of chromosome centromere distribution in co-centric nuclear shells in myoblasts (24 hrs after cell plating) and in differentiated myocytes (after 7 days in a medium containing 2% horse serum).

Microarray Assay To study changes in the global transcriptome Carfilzomib and to correlate the observed centromere repositioning with genes being switched-on/off, we used microarray gene expression analysis. Among the investigated genes, there were 249 transcripts with a 2-fold change in expression in the differentiated myogenic cells compared to the myoblasts.