TP53 negative cancers [80]. However, nearly 70% of all cancers express wt TP53, and demonstrate competence for DNA damage- associated TP53 signalling [81]. Inhibition of the components identified here may be a suitable therapeutic strategy to compromise radiation survival of such cancers. The participation of TP53, p21Cip1/Kip1 and the RB proteins in G1 checkpoint control is recognized [79]. The identification of druggable kinases required for IR-mediated RB1 activation reported here, to our knowledge, represents the first systematic approach towards discovery of targets for the manipulation of DNA damage- associated G1 checkpoint activation. Materials and Methods Cell lines and Antibodies IR was delivered using an AGO HS-MP1 x-ray set. Green fluorescent protein (GFP)-tagged G1 cell cycle reporter encoding the sub-cellular localization domain of human helicase B [47] was purchased from GE Healthcare. CellTiter-GloH cell viability assay was from Promega. Antibodies used were as follows; anti-RB1-P- S608 (Serotec), anti-RB1-PS795 (Sigma), anti-RB1 (Pharmingen), anti-TP53 (NeoMarkers), anti-p21CIP1/WAF1 (Upstate), anti- GAPDH (Abcam), anti-ß-actin (Abcam), Chk1 G-4 sc8408 (Santa Cruz), P-S296 Chk1 2349 S (Cell Signaling), P-S780 Rb 1182-1 (Epitomics). Secondary anti-mouse/rabbit Alexafluor 488/647 (Invitrogen) and secondary HRP-coupled anti-mouse/rabbit IgG (PIERCE). HCT116 human colorectal cancer cells were obtained from ATCC. HCT116 with stable expression of a GFP-tagged cell cycle reporter were generated in house. HCT116 p53+/+ and p532/2 isogenic cell lines were provided by the Vogelstein laboratory. SAR020106 was obtained from Dr. Michelle Garrett, Institute of Cancer Research and used at 1 mM, KU-55933 was obtained form Tocris Bioscience and used at 10 mM. Protein analysis Cell lysates were prepared in HBS buffer (50 mM HEPES pH 7.0, 250 mM NaCl, 0.2% Triton-X100, 1 mM DTT, 1 mM EDTA, 1 mM NaF, 10 mM ß-glycerophosphate, 0.11mM NaVO4 plus EDTA free protease inhibitors (Roche). SDS- polyacrylamide gel electrophoresis followed standard procedures. Proteins were transferred onto PVDF membranes (Millipore) then probed with antibodies and HRP-coupled secondary antibodies and exposed to ECL PlusTM reagent (GE Healthcare Sciences). Glutathione-S-transferase (GST)-assisted pull downs used full- length GST-fused E2F-1 with unfused GST as a control. Assays were performed as detailed in [37]. RNA analysis RNA was prepared using Trizol (Invitrogen) followed by phenol/chloroform extraction. First strand cDNA synthesis was performed using hexamer random primers (Promega). Quantita- tive PCR (qPCR) based analysis was performed using the Precision qPCR master-mix (PrimerDesign) with Taqman primers (Applied Biosystems). Water was used instead of cDNA as background control. An Applied Biosystems Prism Sequence Detection System was used to measure relative gene expression from each sample. High-throughput siRNA screening assay HCT116 cells were reverse transfected in triplicate sets of 96-well PackardView plates (Thermofisher) with siRNA from a kinome- covering library (Dharmacon) in a one-gene, one-well format. Cells were seeded at 8,000 cells/well and transfected using HiPerFect lipid transfection reagent (Qiagen) at a fixed siRNA concentration of 20 nM. Cells were exposed to 5 Gy IR 24 hours following Mechanism of G1 Radiation Checkpoint Activation transfection and fixed at 48 hrs using 3.7% formaldehyde/PBS (Sigma) for 10 min. Fixed cells were permeabilised using 0.1% Triton X-100 (Sigma) in TRIS-buffered Saline (TBS), blocked in 5% skimmed milk powder in TBS/0.1% Tween-20 (Sigma) and probed with anti-RB1-PS780 rabbit monoclonal antibody (Epi- tomics) followed by Alexafluor 488 secondary antibody containing 2 mM Hoechst 33258, diluted in 5% skimmed milk powder in TBS/0.1% Tween-20. The intensity of fluorescent nuclear signal in a minimum of 1500 individual cells was determined using an IN Cell Analyzer 3000 high content image analyzer (GE Healthcare). Data were exported into Microsoft Excel, which was used for data handling and analysis. Intensity profiles were established for individual cell data and gated for the percentage of cells displaying low levels of RB1-PS780 loss (POS-LoRBPS780 value – see Figure 4A). All plates contained triplicate positive controls (p21CIP1/WAF1 siRNA) and triplicate Mock controls (lipid only). Z- factor calculations relating to these controls were applied to evaluate the quality of all plates. [82]. Plate sets were rejected and rerun where any yielded z-prime ,0.2. G1 reporter assay and p21CIP1/WAF1 positivity HCT116 cells stably expressing a green fluorescent protein (GFP)-tagged G1 cell cycle reporter were generated by transfection of the reporter alongside a puromycin encoding plasmid (pBabe- Puro). Transfected cultures were subjected to puromycin selection followed by fluorescence activated cell sorting for GFP positivity. siRNA transfection, treatment and fixation were as for screening. Cells were imaged to determine the G1 cell cycle component by determining the nuclear and cytoplasmic GFP signal ratio for a given cell. At a minimum 1500 individual cells were imaged for each data point. The calculated ratio of cytoplasmic: nuclear fluorescence was gated and the percentage of cells per well determined that had a signal intensity ratio .2, (POS-G1 value), see (Figure S4). To quantify p21CIP1/WAF1 positivity, cells were stained with anti p21CIP1/WAF1 primary antibody, followed by Alexafluor 488 secondary antibody. Nuclear fluorescence for .1500 cells was determined, and cells with objective p21CIP1/WAF1 positivity (POS- p21) identified by gating, see (Figure S4) Cell survival assay HCT116 cells were reverse transfected with siRNA and divided three ways into triplicate plates at a seeding density of 2,666 cells/ well. A plate at a seeding density of 8000 cells/well for determining POS-LoRBPS780 POS was generated in parallel. 24 hours following transfection plates were irradiated with 5 Gy IR, 2 Gy IR or left untreated. Plates for survival assessment were incubated for a further 5 days. The amount of viable cells per well was assessed using CellTiter-GloH. Plates for POS-LoRBPS780 assessment were fixed and processed as for the screen. In addition to silencing the various targets we included siRNA duplexes targeting PLK1, a gene previously shown as being required for viability of Ras-transformed cells [83], to provide a positive control for detecting viability loss. Statistics Z-prime calculations were done using 12(3(sp+sn)/(mp2mn) with p = plate internal positive control or library candidate siRNA, n = plate internal negative controls, s = standard deviation, m = mean. All data are expressed as normalized means 6 SD from at least three independent experiments unless otherwise stated. Z-scores, describing the distance from the target mean to the population mean in units of the standard error, were calculated using standard Z-test statistics. Gene clustering was performed using the heatmap function in ‘R’ statistical package (http://www. PLoS ONE | www.plosone.org 11 February 2012 | Volume 7 | Issue 2 | e31627