combination with siRNA targeting CHK1. siRNA targeting p21CIP1/WAF1 and non-targeting (NT) oligonucleotides were run alongside for control. Cells were irradiated with 2 or 5 Gy or left untreated and viable cells were quantified 5 days following IR exposure. Data are normalized to the respective untreated controls. Open triangles = Mock (Li/NT), open squares = CHK1 only (Li/CHK1), filled triangles = target only (target/NT), filled square = combined target and CHK1 knockdown (target/CHK1). Error bars represent the variance from the mean from three technical replicates. F) Modulation of RB1 phosphorylation by target knockdown. Parallel POS-LoRBPS780 analysis was used to verify siRNA performance. G) Statistical analysis: Student t-test for data shown in A–I. H) Treatment interac- tion. Data were assessed for evidence of interaction between radiation and target knockdown. Values represent the degree of net synergism experienced in IR exposed cells in either Mock- perturbed (NT) or CHK1-perturbed background. (TIF) Figure S7 Interaction of p53 perturbation on survival of cells with target knockdown. A–G) Effects of target kockdown on survival of IR exposed cells. HCT116 cells were transfected with target siRNA alone or in combination with siRNA targeting p53. Cells were treated with IR (5 Gy or 2 Gy) or left untreated (control). Viable cells were quantified 5 days after References 1. Dahele M, Senan S (2010) Radiation oncology: overview and recent advances. J R Coll Physicians Edinb 40: 136–143; quiz 143–134. 2. Elshaikh M, Ljungman M, Ten Haken R, Lichter AS (2006) Advances in radiation oncology. Annu Rev Med 57: 19–31. 3. Kim JJ, Tannock IF (2005) Repopulation of cancer cells during therapy: an important cause of treatment failure. Nat Rev Cancer 5: 516–525. 4. Lindsay KJ, Coates PJ, Lorimore SA, Wright EG (2007) The genetic basis of tissue responses to ionizing radiation. Br J Radiol 80 Spec No 1: S2–6. 5. Jorgensen TJ (2009) Enhancing radiosensitivity: targeting the DNA repair pathways. Cancer Biol Ther 8: 665–670. 6. Kastan MB, Bartek J (2004) Cell-cycle checkpoints and cancer. Nature 432: 316–323. 7. Bao S, Wu Q, McLendon RE, Hao Y, Shi Q, et al. (2006) Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature 444: 756–760. 8. Pauwels B, Wouters A, Peeters M, Vermorken JB, Lardon F (2010) Role of cell cycle perturbations in the combination therapy of chemotherapeutic agents and radiation. Future Oncol 6: 1485–1496. 9. Kinsella TJ (2009) Understanding DNA damage response and DNA repair pathways: applications to more targeted cancer therapeutics. Semin Oncol 36: S42–51. 10. Ward JF (2000) Complexity of damage produced by ionizing radiation. Cold Spring Harb Symp Quant Biol 65: 377–382. 11. Khanna KK, Jackson SP (2001) DNA double-strand breaks: signaling, repair and the cancer connection. Nat Genet 27: 247–254. 12. Lukas J, Lukas C, Bartek J (2004) Mammalian cell cycle checkpoints: signalling pathways and their organization in space and time. DNA Repair (Amst) 3: 997–1007. 13. Liu Q, Guntuku S, Cui XS, Matsuoka S, Cortez D, et al. (2000) Chk1 is an essential kinase that is regulated by Atr and required for the G(2)/M DNA damage checkpoint. Genes Dev 14: 1448–1459. 14. Zhao H, Watkins JL, Piwnica-Worms H (2002) Disruption of the checkpoint kinase 1/cell division cycle 25A pathway abrogates ionizing radiation-induced S and G2 checkpoints. Proc Natl Acad Sci U S A 99: 14795–14800. 15. Chen MS, Ryan CE, Piwnica-Worms H (2003) Chk1 kinase negatively regulates mitotic function of Cdc25A phosphatase through 14-3-3 binding. Mol Cell Biol 23: 7488–7497. 16. Lavin MF (2008) Ataxia-telangiectasia: from a rare disorder to a paradigm for cell signalling and cancer. Nat Rev Mol Cell Biol 9: 759–769. 17. Sanchez Y, Wong C, Thoma RS, Richman R, Wu Z, et al. (1997) Conservation of the Chk1 checkpoint pathway in mammals: linkage of DNA damage to Cdk regulation through Cdc25. Science 277: 1497–1501. 18. Chen Z, Xiao Z, Chen J, Ng SC, Sowin T, et al. (2003) Human Chk1 expression is dispensable for somatic cell death and critical for sustaining G2 DNA damage checkpoint. Mol Cancer Ther 2: 543–548. 19. Uto K, Inoue D, Shimuta K, Nakajo N, Sagata N (2004) Chk1, but not Chk2, inhibits Cdc25 phosphatases by a novel common mechanism. Embo J 23: 3386–3396. Mechanism of G1 Radiation Checkpoint Activation IR. Data are normalized to the untreated controls. Error bars depict the variance from the mean for three technical replicates. Filled square = combined target and p53 knockdown (target/p53) filled triangles = target only (target/NT), open triangles = Mock (Li/NT). H) Modulation of RB1 phosphorylation by target knockdown. Parallel POS-LoRBPS780 analysis, verifying siRNA performance. I, K) Treatment interaction. Data were assessed for evidence of interaction between radiation and target knockdown. Values represent the degree of net synergism experienced in IR exposed cells. Note absence of significant synergy in p53-perturbed backgrounds. (TIF) Table S1 Screen data. Target official gene symbol in alphabetical order; average POS-LoRBPS780 (Average), variation from the mean for n = 3 replicates (Standard Deviat) and Z-score statistics calculated from the average POS-LoRBPS780 (Z-score) are shown for each target. (PDF) Author Contributions Conceived and designed the experiments: SM ER WA SS. Performed the experiments: ER. Analyzed the data: ER SS. Contributed reagents/ materials/analysis tools: HL MEC. Wrote the paper: SM ER. 20. Alderton GK, Galbiati L, Griffith E, Surinya KH, Neitzel H, et al. (2006) Regulation of mitotic entry by microcephalin and its overlap with ATR signalling. Nat Cell Biol 8: 725–733. 21. Reinhardt HC, Yaffe MB (2009) Kinases that control the cell cycle in response to DNA damage: Chk1, Chk2, and MK2. Curr Opin Cell Biol 21: 245–255. 22. Reinhardt HC, Hasskamp P, Schmedding I, Morandell S, van Vugt MA, et al. (2010) DNA damage activates a spatially distinct late cytoplasmic cell-cycle checkpoint network controlled by MK2-mediated RNA stabilization. Mol Cell 40: 34–49. 23. Bunz F, Dutriaux A, Lengauer C, Waldman T, Zhou S, et al. (1998) Requirement for p53 and p21 to sustain G2 arrest after DNA damage. Science 282: 1497–1501. 24. Chan TA, Hwang PM, Hermeking H, Kinzler KW, Vogelstein B (2000) Cooperative effects of genes controlling the G(2)/M checkpoint. Genes Dev 14: 1584–1588. 25. Burkhart DL, Sage J (2008) Cellular mechanisms of tumour suppression by the retinoblastoma gene. Nat Rev Cancer 8: 671–682. 26. Assoian RK, Yung Y (2008) A reciprocal relationship between Rb and Skp2: implications for restriction point control, signal transduction to the cell cycle and cancer. Cell Cycle 7: 24–27. 27. Smith ML, Zhan Q, Bae I, Fornace AJ, Jr. (1994) Role of retinoblastoma gene product in p53-mediated DNA damage response. Exp Cell Res 215: 386–389. 28. Haapajarvi T, Kivinen L, Pitkanen K, Laiho M (1995) Cell cycle dependent effects of u.v.-radiation on p53 expression and retinoblastoma protein phosphorylation. Oncogene 11: 151–159. 29. Harrington EA, Bruce JL, Harlow E, Dyson N (1998) pRB plays an essential role in cell cycle arrest induced by DNA damage. Proc Natl Acad Sci U S A 95: 11945–11950. 30. Slebos RJ, Lee MH, Plunkett BS, Kessis TD, Williams BO, et al. (1994) p53- dependent G1 arrest involves pRB-related proteins and is disrupted by the human papillomavirus 16 E7 oncoprotein. Proc Natl Acad Sci U S A 91: 5320–5324. 31. Lanni JS, Jacks T (1998) Characterization of the p53-dependent postmitotic checkpoint following spindle disruption. Mol Cell Biol 18: 1055–1064. 32. Deckbar D, Stiff T, Koch B, Reis C, Lobrich M, et al. The limitations of the G1- S checkpoint. Cancer Res 70: 4412–4421. 33. Agerbaek M, Alsner J, Marcussen N, Lundbeck F, von der Maase H (2003) Retinoblastoma protein expression is an independent predictor of both radiation response and survival in muscle-invasive bladder cancer. Br J Cancer 89: 298–304. 34. Jahnson S, Risberg B, Karlsson MG, Westman G, Bergstrom R, et al. (1995) p53 and Rb immunostaining in locally advanced bladder cancer: relation to prognostic variables and predictive value for the local response to radical radiotherapy. Eur Urol 28: 135–142. 35. Moonen L, Ong F, Gallee M, Verheij M, Horenblas S, et al. (2001) Apoptosis, proliferation and p53, cyclin D1, and retinoblastoma gene expression in relation to radiation response in transitional cell carcinoma of the bladder. nt J Radiat Oncol Biol Phys 49: 1305–1310. PLoS ONE | www.plosone.org 13 February 2012 | Volume 7 | Issue 2 | e31627