Mechanism of G1 Radiation Checkpoint Activation Figure 1. siRNA screen for gene products involved in IR-associated RB1 activation. A) Loss of RB1 phosporylation in IR exposed cells. HCT116 cells exposed to 5 Gy ionising radiation (IR) or untreated (C) were analysed at the time indicated. Levels of RB1 with phosphorylation on Ser608 (RB1-PS608), total RB1 (RB1) and TP53 were established by immunoblotting. B) Loss of RB1 phosphorylation is TP53-dependent. TP53 positive (+/+) and isogenic TP53 (2/2) HCT116 cells exposed to 5 Gy ionizing radiation (IR) or untreated (C) were analysed 24 hours post irradiation. Levels of RB1-PS608, total RB1 and p21CIP1/WAF1 were established by immunoblotting. C) IR affects RB1 phosphorylation at multiple sites. Immunoblots probing for levels of RB1-PS608, RB1-PS780, RB1-PS795 in irradiated (IR) or untreated (C) HCT116. To document RB1 specificity of the signal cells transfected with siRNA duplexes targeting RB1 (RB(1) and RB(2)) or nontargeting control siRNA (NT) were analysed in parallel. Cells were irradiated and harvested at 24 hours following IR. Actin was used as a loading control. D) siRNA screening strategy. HCT116 were reverse transfected with siRNA library pools in a 96 well format, and irradiated, fixed and stained using anti RB1-PS780 antibody and Hoechst 33342 dye, with timelines as indicated. Plates were analysed using an IN Cell Analyzer 3000 high content platform (GE) with sequential blue and green laser excitation. A set number of cell objects per well were analyzed for nucleus-associated antibody fluorescence (green channel). Hoechst 3342 DNA staining (blue channel) was used for object and compartment identification. Intensity profiles were generated and automatically gated to determine the percentage of cells with sub-normal antibody fluorescence (POS-LoRBPS780) in individual wells. E) Radio-resistant RB1 phosphorylation in cells with siRNA-mediated TP53-signalling knockdown. Assay set up was as described in D, siRNA pools for TP53, p21CIP1/WAF1 or a non-targeting oligonucleotide (nt) were used for transfection. Error bars relate to variance in POS-LoRBPS780 values from triplicate wells. F) Primary screen outcome. Z-score distribution for target screened. Z-scores were calculated for the mean POS-LoRBPS780 observed in triplicate wells and are plotted in ranked order. Hits are shown colour-coded according to hit class within the Z-score distribution. doi:10.1371/journal.pone.0031627.g001 When re-examined in this way, half of the strong hits (6 of 12) and two hits from the weaker category confirmed with two or more oligonucleotides (Figure 2C), together yielding 8 hits validating with multiple oligonucleotides, representing the p53-related protein kinase PRPK/TP53RK, the mammalian sterile 20-like MAPK pathway component serine threonine kinase STK4/MST1, the cyclin dependent kinase CDK4, the dual specificity tyrosine (Y)- phosphor- ylation-regulated kinase DYRK1A, the glucose-phosphorylating, glycolytic enzyme hexokinase HK1, the cyclic AMP- dependent protein kinase, gamma catalytic subunit PRKACG and p21CIP1/WAF1/CDKN1A. Real-time PCR (RT-PCR) analysis (Figure S2) showed that treatment with the respective oligonucleotides led to transcript knockdown in all instances. Corroborating our original analysis, DAVID analysis confirmed representation of MAPK (STK4, and PRKACG) and calcium signalling components (PRKACG) amongst the validated hits, as well as representation of hits that do not group to the annotated pathway ontology (CDK4, DYRK1A, HK1, p21CIP1/WAF1, PRPK). Effect of target knockdown on IR-mediated p21CIP1/WAF1 expression To explore how the various hits contribute to the radiation response we examined the effects of their knockdown on the IR- induced accumulation of p21CIP1/WAF1. As mentioned previously, PLoS ONE | www.plosone.org 3 February 2012 | Volume 7 | Issue 2 | e31627