Replicative cellular senescence is an important tumor suppression mechanism and also contributes to aging. open, and the transcription of satellite sequences increases. The peripheral heterochromatic compartment (PHC) becomes less prominent, and centromere structure becomes notably enlarged. These epigenetic changes progress slowly after the onset of senescence, with some, such as mobilization of retrotransposable elements, becoming prominent only at late times. Many of these changes have also been noted in cancer cells. Introduction Replicative cellular senescence was first described as an irreversible growth arrest triggered by the accumulation of cell divisions (Hayflick & Moorhead, 1961). Subsequently it has emerged as a potent tumor suppression mechanism, and recent evidence points to important connections with aging (Collado in tissues as well as in cell culture during replicative senescence, and that this occurs largely in repetitive DNA sequences (Sedivy (Day (Coufal et al., 2009). Primer design is described below, and all primers are listed in Table S1. For qPCR of DNA, purified genomic or FAIRE DNA was used with the indicated primers. For qPCR of RNA, total RNA was harvested from cells using Trizol reagent (Invitrogen) according to the manufacturers instructions. 1 g of total RNA was transcribed into cDNA in 50 l reactions using the Taqman kit (Applied Biosystems), according to the manufacturers protocol. 1 l of this reaction was used in subsequent qPCR reactions. GAPDH was used as the normalization control. For the measurement Rabbit Polyclonal to OR2AG1/2 repetitive DNA transcription, total RNA was exhaustively digested with RNase free DNase, and further cleaned up on RNeasy columns, SB-207499 prior to the synthesis of cDNA. Effectiveness of the DNase digestion was assessed using controls that omitted reverse transcriptase. Design of PCR primers See Table S1 for a listing of all primers. For detailed methods, see Supplemental Information. Chromatin immunoprecipitation All procedures followed the protocols in the Magna ChIP kit (Millipore). For a detailed protocol, see Supplemental Information. Electron microscopy Cells were grown in 10 cm dishes as indicated. For a detailed protocol, see Supplemental Information. Fluorescence in situ hybridization Cells were grown on coverslips, fixed with 4% paraformaldehyde for 20 min at room temperature, washed 3x in SB-207499 phosphate buffered saline (PBS), and stored in 70% ethanol at 4C until used. For a detailed protocol, see Supplemental Information. Supplementary Material Supplementary dataClick here to view.(14M, pdf) Acknowledgments P.D. Adams and his lab (Beatson Institute) are gratefully acknowledged for a productive ongoing collaboration and communication of unpublished data and reagents. This work was supported by NIH/NIA grant R37 AG016694 to J.M.S. M.D.C. was supported in part by Dottorato di Ricerca in Biotecnologie, Farmacologia e Tossicologia, PFDR in Biotecnologie Cellulari e Molecolari fellowship of Bologna University. S.W.C. was supported in part by NIH/NIGMS Institutional Research Training Grant T32 GM007601. J.A.K. was supported in part by NIH/NCRR grant P20 RR015578-10S1 and a Mentored Research Scientist Development Award from the NIH/NIA K01 AG039410. N.N. was supported by a Mentored SB-207499 Quantitative Research Development Award from the NIH/NIA K25 AG028753 and K25 AG028753-03S1. The Genomics Core Facility in the Laboratories for Molecular Medicine at Brown University was supported in part by the COBRE award from the NIH/NIGMS P30 GM0103410. J.M.S. is a Senior Scholar of the Ellison Medical Foundation and a recipient of the Glenn Award for Research on the Biological Mechanisms of Aging from the Glenn Medical Foundation..