FBS. Apoptosis was determined by detection of the externalization of phosphatidylserine in the inner towards the outer leaflet of the plasma membrane together with the annexin V ITC detection kit (Roche Biosciences) and flow cytometry (FACSCalibur, Becton Dickinson) (56). Apoptosis was also quantified in U2OS cells and fl/fl or / MEFs with an enzyme immunoassay (Roche Applied Science) using a combination of antibodies that recognize histones and DNA, as described previously (57). Apoptosis was also determined by detecting the amounts of cleaved PARP and cleaved caspase-3 in cell lysates by Western blotting. Cell proliferation was measured together with the MTT assay as described previously (47).NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptSci Signal.Methyl 6-aminopicolinate manufacturer Author manuscript; out there in PMC 2014 July 23.Xu et al.PageImage quantification and statistical analysis Pictures were quantified with ImageJ software. Information are presented as indicates ?SEM from no less than three independent experiments. P values were calculated by two-tailed Student’s t test or one-way ANOVA with Tukey’s post hoc test working with Minitab 13 software program (Minitab).NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptSupplementary MaterialRefer to Net version on PubMed Central for supplementary material.AcknowledgmentsWe thank A. Resnick, M. Saleh, R. Tyagi, M. Ma, J. Sbodio, R. Tokhunts, R. Mealer, P. Scherer, M. Harraz, and also other members with the Solomon H. Snyder laboratory for helpful discussions. We thank B. Ziegler for organizing the manuscript. Funding: This perform was funded by the U.S. Public Well being Service grant DA-000266 plus the Medical Scientist Instruction Program T32 grant (to R.X.).REFERENCES AND NOTES1. Vousden KH, Lu X. Live or let die: The cell’s response to p53. Nat Rev Cancer. 2002; two:594?04. [PubMed: 12154352] 2. Chen Z, Trotman LC, Shaffer D, Lin HK, Dotan ZA, Niki M, Koutcher JA, Scher HI, Ludwig T, Gerald W, Cordon-Cardo C, Pandolfi PP. Vital function of p53-dependent cellular senescence in suppression of Pten-deficient tumorigenesis. Nature. 2005; 436:725?30. [PubMed: 16079851] 3. Collado M, Gil J, Efeyan A, Guerra C, Schuhmacher AJ, Barradas M, Bengur A, Zaballos A, Flores JM, Barbacid M, Beach D, Serrano M. Tumour biology: Senescence in premalignant tumours. Nature. 2005; 436:642. [PubMed: 16079833] four. Bartkova J, Horejs?Z, Sehested M, Nesland JM, Rajpert-De Meyts E, Skakkebaek NE, Stucki M, Jackson S, Lukas J, Bartek J. DNA harm response mediators MDC1 and 53BP1: Constitutive activation and aberrant loss in breast and lung cancer, but not in testicular germ cell tumours.N-Methyl-3-phenylpropan-1-amine manufacturer Oncogene.PMID:33740179 2007; 26:7414?422. [PubMed: 17546051] 5. Voorhoeve PM, le Sage C, Schrier M, Gillis AJ, Stoop H, Nagel R, Liu YP, van Duijse J, Drost J, Griekspoor A, Zlotorynski E, Yabuta N, De Vita G, Nojima H, Looijenga LH, Agami R. A genetic screen implicates miRNA-372 and miRNA-373 as oncogenes in testicular germ cell tumors. Cell. 2006; 124:1169?181. [PubMed: 16564011] 6. Wade M, Li YC, Wahl GM. MDM2, MDMX and p53 in oncogenesis and cancer therapy. Nat Rev Cancer. 2013; 13:83?6. [PubMed: 23303139] 7. Drost J, Mantovani F, Tocco F, Elkon R, Comel A, Holstege H, Kerkhoven R, Jonkers J, Voorhoeve PM, Agami R, Del Sal G. BRD7 is usually a candidate tumour suppressor gene necessary for p53 function. Nat Cell Biol. 2010; 12:380?89. [PubMed: 20228809] eight. Odom AR, Stahlberg A, Wente SR, York JD. A part for nuclear inositol 1,4,5-trisphosphate kinase in transcriptional control. Science. 2000;.