two additional cell types using different ways to activate p53: a p19ARF-induced cell cycle arrest and a DNA damage mediated cell cycle arrest. We conclude that ARNTL suppression affects the ability of p53 to induce a cell cycle arrest upon cellular stress signals such as DNA damage. Materials and Methods Cell lines & culture conditions BJtsLT cells were cultured in medium that consisted of DMEM 75% / M199 25% supplemented with 10% FCS, Penicillin and Glutamine. BJtsLT cells were cultured at 32uC in 5% CO2. U2OS and Phoenix cells were cultured in DMEM supplemented with 10% FCS, Penicillin and Glutamine. U2OS and Phoenix cells were cultured at 37uC in 5% CO2. Plasmids and library Expression plasmid for ARNTL was generated by PCR from a cDNA library and subsequent cloning the PCR product into pCR3-Flag. The P19-RFP construct was described previously [11]. The construction of the library was described previously [11]. Briefly, the NKI shRNA library was designed to target 7914 human genes, using three shRNA vectors for every targeted gene, cumulating in a total of 23,742 shRNA vectors. The shRNAs are cloned into a retro-viral vector to enable infection of target cells. Retroviral infection Phoenix cells were transfected using calcium phosphate method. Viral supernatant was cleared through a 0.45 mM filter. Cells were infected with the viral supernatant in presence of polybrene (8 mg/ ml). The infection was repeated twice. shRNA barcode screen To screen the NKI shRNA library we reasoned that we would need 100 fold coverage of the library to get a good representation of all 23,742 shRNA vectors present in the library. BJtsLT cells were infected with the NKI shRNA library. Two days after infection cells were plated at 150,000 cells/15 cm dish. In total 26106 cells were shifted to 39uC, equal number of cells were kept at 32uC. Cells at 32uC were harvested after 5 days. Cells at 39uC were harvested at 21 days after shift. From both populations gDNA was isolated using DNAzol (Invitrogen). The shRNA cassettes were amplified by PCR. The PCR product was used for in vitro RNA synthesis. RNA was labeled with Cy3 or Cy5 (Kreatech) and hybridized on a microarray. Quantification of the resulting fluorescent images was performed with Imagene 5.6 (BioDiscovery), local background was subtracted, and the data were normalized and 2log transformed. Additional information on barcode screens can be found at http://www.screeninc.nl. References 1. Vousden KH, Lane DP (2007) p53 in health and disease. Nat Rev Mol Cell Biol 8: 275–283. 2. Toledo F, Wahl GM (2006) Regulating the p53 pathway: in vitro hypotheses, in vivo veritas. Nat Rev Cancer 6: 909–923. 3. Chuikov S, Kurash JK, Wilson JR, Xiao B, Justin N, et al. (2004) Regulation of p53 activity through lysine methylation. Nature 432: 353–360. 4. Brooks CL, Gu W (2006) p53 ubiquitination: Mdm2 and beyond. Mol Cell 21: 307–315. 5. Brooks CL, Gu W (2003) Ubiquitination, phosphorylation and acetylation: the molecular basis for p53 regulation. Curr Opin Cell Biol 15: 164– 171. 6. Gu W, Roeder RG (1997) Activation of p53 sequence-specific DNA binding by acetylation of the p53 C-terminal domain. Cell 90: 595–606. 7. Momand J, Zambetti GP, Olson DC, George D, Levine AJ (1992) The mdm-2 oncogene product forms a complex with the p53 protein and inhibits p53- mediated transactivation. Cell 69: 1237–1245. shRNA Screen Modulators of p53 Colony formation assay Cells were infected with retroviral supernatant. Two days after infection the cells were seeded at 50,000/10 cm dish and shifted to 39uC. Cells were cultured at 39uC for approx 21 days. When colonies appeared cells were fixed in MeOH/HAc (3:1) and subsequently stained (50% MeOH/10% HAc/0.1% Coomassie). Western blotting Cell lysates were separated using 10% SDS-PAAGE. Proteins were transferred to PVDF membrane and incubated with primary antibody as indicated. Primary antibodies were detected using a secondary HRP-conjugated antibody. Antibodies used for these studies: Flag (M2; Sigma), TNIP1 (1A11E3; Zymed), CDK4 (C- 22; Santa Cruz), p53 (DO-1; Santa Cruz) and p21CIP1 (C-19; Santa Cruz). QRT-PCR Total RNA was isolated using TRIzol (Invitrogen). From the total RNA cDNA was generated using Superscript II (Invitrogen) using random primers (Invitrogen). cDNA was diluted and QRT reaction was performed using Taqman probes (Applied Biosys- tems). All QRT reactions were run in parallel for GAPDH to control of for input cDNA. The QRT reactions were run at a AB7500 Fast Real Time PCR system (Applied Biosystems). Results shown are a representative of three independent experiments. p21CIP1 induction by c-radiation and PXD101 For the p21CIP1 induction by radiation 50,000 cells were seeded per 6-well. HCT116 p532/2 cells were irradiated with 20 Gy c- radiation from a Cs-137 source. Cells were incubated for 16 hrs and lysed. For the p21CIP1 induction by PXD101 50,000 cells were seeded/6-well and treated with 0.5 mM PXD101 for 16 hrs after which the cells were lysed and protein lysates were subjected to western analysis. Acknowledgments We thank K Berns and EM Heijmans for the BJtsLT cells. In addition, we thank all members of the Beijersbergen and Bernards labs for helpful discussions. Author Contributions Conceived and designed the experiments: JM AWF RB RB. Performed the experiments: JM AWF MM. Analyzed the data: JM AWF RB. Wrote the paper: JM AWF RB RB. Performed the microarray hybridizations: MM. Supervised the research and corrected the manuscript: RB RLB. 8. Riley T, Sontag E, Chen P, Levine A (2008) Transcriptional control of human p53-regulated genes. Nat Rev Mol Cell Biol 9: 402–412. 9. http://www-p53.iarc.fr/ IARC p53 Database. 10. Oliner JD, Kinzler KW, Meltzer PS, George DL, Vogelstein B (1992) Amplification of a gene encoding a p53-associated protein in human sarcomas. Nature 358: 80–83. 11. Berns K, Hijmans EM, Mullenders J, Brummelkamp TR, Velds A, et al. (2004) A large-scale RNAi screen in human cells identifies new components of the p53 pathway. Nature 428: 431–437. 12. Berns K, Horlings HM, Hennessy BT, Madiredjo M, Hijmans EM, et al. (2007) A functional genetic approach identifies the PI3K pathway as a major determinant of trastuzumab resistance in breast cancer. Cancer Cell 12: 395–402. 13. Brummelkamp TR, Fabius AW, Mullenders J, Madiredjo M, Velds A, et al. (2006) An shRNA barcode screen provides insight into cancer cell vulnerability to MDM2 inhibitors. Nat Chem Biol 2: 202–206. PLoS ONE | www.plosone.org 9 March 2009 | Volume 4 | Issue 3 | e4798