Human SAHFs contain several common markers of heterochromatin as CBX1, known as HP1beta, macroH2A and HMGA. It has been shown that each SAHF in senescent cells results from condensation of an individual chromosome. The earliest detectable event in the LY294002 Abmole Mechanosensitive caveolin-1 activation-induced PI3K/Akt/mTOR signaling pathway promotes breast cancer motility, invadopodia formation and metastasis in vivo formation of a SAHF focus is the chromosome condensation, followed by methylation of lysine 9 of histone H3, binding of HP1 proteins and incorporation of macroH2A. In the present work we have studied constitutive heterochromatin distribution in bovine Abmole LY294002 cultured fibroblasts that reached proliferative senescence at late in vitro passages. We detected heterochromatin domains using a BAC probe specific for pericentric chromosomal regions of all bovine autosomes and using antibodies specific for histone H3 three-methylated at lysine 9 that is enriched in heterochromatic chromosomal domains. We also performed immunodetection of 5-methyl cytosine, CENP A/ B as well as counterstaining with DAPI and YoPro1. We did not reveal any SAHF-like structures in senescent bovine fibroblasts. Instead, we observed fibrous distribution of constitutive heterochromatin that formed ribbon-like and ring-like structures associated with the nucleolar periphery. Bovine primary fibroblasts were cultured in vitro for 25�C34 passages. Replicative senescence of bovine fibroblasts was determined by terminal growth arrest, morphological changes, i.e. an increase in cell size, an irregular shape, dark cytoplasmic granules and the proportion of senescent-associated beta-galactosidase-positive fibroblasts in non-confluent cultures. In order to detect senescence, we have stained cells with beta-galactosidase at each passage. Typical staining pattern is shown in Figure S1A. Next we have counted the proportion of beta-galactosidase-positive cells at each passage. The quantity of positively stained cells increased during passages in agreement with published data. Starting from passage 24, cell cultures consisted of.95% cells positively stained with beta-galactosidase assay and had enlarged morphology. Cells that reached senescence could be cultivated up to passage 34 and be kept in culture for up to 8 weeks without apoptosis. The clusters of heterochromatin observed in young cells correspond to chromocenters. Indeed, these heterochromatic domains detected by antibodies against H3K9me3 were always associated with chromosomal centromeres when co-detection for CENP A/B proteins was performed. Moreover, heterochromatin clusters were clearly observed by FISH with the BAC231G08 probe specific for pericentric chromosomal domains of autosomes. On the other hand, using these two approaches we did not detect true chromocenters in senescent cells.