Mechanism of G1 Radiation Checkpoint Activation phorylation (Figure 1E an d 2C) supports the critical role of this gene in G1 checkpoint activation. We therefore hypothesized that knockdown of at least some of the targets identified act by affecting p21CIP1/WAF1 accumulation. To test this hypothesis, we adapted the method for quantifying antibody fluorescence for assessment of p21CIP1/WAF1 abundance. To determine the percentage of p21CIP1/WAF1-positive cells (POS- p21) we gated for nuclear signal intensity substantially higher than the background fluorescence in cells with ablation of the transcription regulator TP53, known to facilitate p21CIP1/WAF1 induction in irradiated cells [45] (Figure S4 and Material and Methods). As expected IR treatment of cells led to a robust increase in the percentage of cells with p21CIP1/WAF1 positivity at 16 hrs, the time when RB1 activation is first apparent, in either Mock transfected cells or cells transfected with NT oligonucleotide (Figure 3). A substantial and highly significant reduction in the percentage of p21CIP1/WAF1 positive cells was seen upon knockdown of three of the validated targets, PRPK/TP53RK, the MAPK pathway component STK4/MST1 and CDK4 (Figure 3A, C). Notably, knockdown of the remaining three targets, DYRK1A, HK1, and PRKACG, had minor and non- significant effects (Figure 3A, C), although their knockdown effectively prevented IR-induced loss of RB1 phosphorylation in a parallel assessment (Figure 3B). Figure 3. Effect of target knockdown on IR-mediated p21CIP1/WAF1 induction. A) Effect of target knockdown on p21CIP1/WAF1 positivity. HCT116 cells transfected with siRNA as indicated were irradiated (IR) or left untreated (control). Cells were assessed for p21CIP1/WAF1 positivity 16 hrs post IR. The percentage of cells with p21CIP1/WAF1 positivity relative to Mock-treatment (Lipid) is shown. Error bars represent the variance of the mean of three biological replicates, run in triplicate. B) Modulation of RB1 phosphorylation by target knockdown. POS- LoRBPS780 analysis was performed in parallel plates. Data points represent the means of triplicate technical replicates and are evaluated using hit classification as for the screen. C) Statistical analysis. Paired t-tests results for data shown in A. D) Treatment interaction test. Targets that yielded significant impairment of p21CIP1/WAF1 positivity were tested for evidence of interaction between radiation and target knockdown. Values indicate the degree of antagonism experienced in IR exposed cells. doi:10.1371/journal.pone.0031627.g003 PLoS ONE | www.plosone.org 5 February 2012 | Volume 7 | Issue 2 | e31627