Knockdown of PRPK and STK4 also reduced p21CIP1/WAF1 positivity in the unirradiated cells (Figure 3A, C), indicating the potential involvement of these kinases in signalling contexts independent of IR challenge. Mathematical testing for an interaction between knockdown of these targets and irradiation (see Materials and Methods) provides evidence for a net antagonism of target knockdown with IR-mediated p21CIP1/WAF1 accumulation that is not explained by the reduction of p21CIP1/WAF1 expression in unchallenged cells (Figure 3D). Hence although PRPK and STK4 promote p21CIP1/WAF1 positivity in an IR-independent context, these genes in addition have a significant role in supporting a DNA damage-associated increase in p21CIP1/WAF1 positivity. Together our experiments suggest a significant involvement of a group of the hits in facilitating p21CIP1/WAF1 positivity upon irradiation. In contrast other hits must play a role in radiation mediated RB1 activation unconnected to p21CIP1/WAF1positivity. Effects on IR-mediated G1 arrest Since DNA damage-induced activation of RB1 is thought to promote cell cycle arrest in G1 [29,46] we tested if the identified Mechanism of G1 Radiation Checkpoint Activation hits are required for this response. To assess cell cycle response we used a GFP-tagged cell-cycle reporter that localizes to the nucleus during G1 but redistributes to the cytoplasm as a consequence of CDK2 activation and S phase entry [47]. Using HCT116 cells with stable expression of this reporter we determined the percentage of G1 cells following IR exposure and upon knockdown of the various hits (Figure 4). Cells with a ratio of nuclear to cytoplasmic fluorescence of two or greater were considered G1 (POS-G1, Figure S4 and Materials and Methods). As previously, we used POS-LoRBPS780 analysis alongside this assessment to monitor for siRNA performance (Figure 4B). Knockdown of all targets led to loss of G1 cells when compared to Mock (Lipid) treatment or treatment with NT oligonucleotide. The cumulative data scored significantly in paired Student’s t-tests in all instances except DYRK1A where, however, the calculated p- value (0.08) strongly converged towards significance (Figure 4C). None of the targets when knocked down caused significant changes in the G1 content in non-irradiated cells (Figure 4A, C), indicating the encoding genes do not act by affecting normal cell cycle progression. Mathematical testing for interaction between Figure 4. Effect of target knock down on G1 checkpoint activation. A) Effect of target knockdown on relative G1 positivity. HCT116 cells transfected with siRNA as indicated were irradiated (IR) or left untreated (control). Cells were fixed 16 hours later and assessed for the proportion of cells in G1. The degree of G1 positivity relative to Mock-treated (Lipid) cells is shown. Error bars represent the variance from the mean of three biological replicates, run in triplicate. B) Modulation of RB1 phosphorylation by target knockdown. POS-LoRBPS780 analysis performed in parallel to A). Data points represent the means of triplicate technical replicates. C) Statistical analysis. Paired t-tests for data shown in A. D) Treatment interaction test. Data were assessed for evidence of a interaction between radiation and target knockdown. Values indicate the degree of antagonism experienced in IR exposed cells. doi:10.1371/journal.pone.0031627.g004 PLoS ONE | www.plosone.org 6 February 2012 | Volume 7 | Issue 2 | e31627