Rproject.org), using the normalized means from three individual experiments for input. Data from the p21CIP1/WAF1 analysis and G1 reporter assays were tested using Student’s paired t-test. Tests for interaction between target knockdown and treatment were performed as described [84]. Briefly, the individual effect of target knockdown and treatment was considered. The impact of target knockdown in the absence of IR (Rc) compared to Mock knockdown in the absence of irradiation (Cc) is designated Rc/Cc. The impact of irradiation on Mock-transfected cells is designated CIR/Cc. From these the expected combined response of target knockdown and IR is derived by (Rc/Cc*Cx/Cc). The degree (index) of interaction, either positive (sensitization) or negative (antagonism), is calculated by subtracting the observed combined effect of IR and target knockdown Rx/Cc form the expected interaction, (Rc/Cc*CIR/Cc)2(RIR/Cc), where C = Mock-transfected, R = target RNAi tansfected, IR = irradiated, c = untreated. An interac- tion is considered antagonistic if the effect in CIR exceeds that in RIR, and synergistic when the effect in RIR exceeds that in CIR. Supporting Information Figure S1 Modification of RB1 activity by IR. A), A9) Signal quantification for results in Figure 1A. Charts depict raw background corrected signal for P-S608 RB1 or relative signal intensity relative to that of pan RB1 in the same samples. Quantification was performed using electronic scans produced from primary autoradiograms. Data were analysed using ImageJ (http://rsbweb.nih.gov/ij/). B), B9) Signal quantification for results in Figure 1B. Charts depict raw background corrected signal for P-S608 RB1, or P-S608 RB1 signal relative to that of pan RB1 in the same samples. Quantification and analysis was performed as in A. C) IR activates RB1 E2F-binding capacity. Lysates from IR treated (IR) and control (C) HCT116 cells were incubated with GST-E2F-1 or unfused GST proteins, coupled to Glutathione-Sepharose beads. Material retained on the beads was probed for the presence of RB1 using immunoblotting. Immunoblot analysis of input lysate indicating reduced RB1-PS780 in IR exposed cells. Note increased amount of RB1 signal in pull-down from IR exposed cells. D) Effect of gene knockdown on RB1 phosphorylation in irradiated and control cells. Z-scores for POS-LoRBPS780 in untreated and irradiated cells. (TIF) Figure S2 Cellular response to target inhibition. A) IR dependent RB1 activation following pharmacological inhibition of double stand break signalling. HCT116 cells seeded in 96 well dishes were treated with CHK1 selective inhibitor SAR020106 (1 mM) or the ATM/ATR selective inhibitor KU-55933 (10 mM) for 5 hrs prior to exposure to as indicated. Transfection with siRNA for p53 served as a positive control. NT denotes transfection with NT oligonucleotide, MOCK defers to mock transfected cells. Plates were processed for assessment 24 hrs post IR as for Figure 1E. B) IR dependent CHK1 activation following inhibition of double strand break signalling. HCT116 seeded in 6 well dishes and treated in parallel to A) were lysed and analysed for CHK1 autophos- phorylation activity. C) Signal quantification for results in Figure S2B. Charts depict background corrected signal for P- S296 CHK1 relative to pan CHK1 in the same samples. Signal detection involved infrared fluorophore-coupled secondary anti- bodies with signal quantification using a Li-COR Odyssey infrared imager. D) IR dependent RB1 phosphorylation change following pharmacological inhibition of double strand break signalling. Levels of Ser780 phosphorylated RB1 (RB1- Mechanism of G1 Radiation Checkpoint Activation P-S780) and total RB1 (RB1) were established 16 hrs post irradiation by immunoblotting. E) Signal quantification for results in Figure S2D. Charts depict background corrected signal for P-S780 CHK1 relative to pan RB1 in the same samples. Signal detection and quantification was as for Figure S2C. F) Active siRNA species deplete target mRNA in transfected cells. HCT116 cells were transfected with single siRNA oligonu- cleotides as indicated and treated with 5 Gy of IR. RNA was isolated 16 hrs post IR exposure. Transcripts were quantified using Taqman RT/qPCR. Data were normalized against GAPDH. Levels relative to those in cells transfected with NT siRNA are shown. Error bars represent the variance from the mean of triplicate technical replicates. Genes analysed were CDK4, DYRK1A, HK1, SPHK2, STK4, PRPK or p21CIP1/WAF1. (TIF) Figure S3 Effect of double stand break signalling inhibition on G1 checkpoint activation. HCT116 cells seeded in 96 well dishes were treated with CHK1 selective inhibitor SAR020106 (1 mM) or the ATM/ATR selective inhibitor KU-55933 (10 mM) for 5 hrs prior to exposure to IR. Transfection with siRNA for p53 served as a positive control. NT denotes transfection with NT oligonucleotide, MOCK refers to mock transfected cells. Data shown are derived though multiplex analysis of experiments shown in Figure S2A. Data assessment was as for Figure 4A. (TIF) Figure S4 Fixed-cell-assay data evaluation methodolo- gy. A) POS-LoRBS780, determining the fraction of cells with low RB1-PS780 signal relative to the total number of cells measured. B) POS-p21, determining the fraction of cells with objective p21CIP1/WAF1 positivity relative to the total number of cells measured. C) POS-G1, determining the fraction of cells with objective G1 positivity relative to the total number of cells measured. Data evaluation relied upon gating for responders based on histogram differences between negative (non-targeting) and positive control (control target), run within the same plate. Example positive (ve+) and negative (ve-) histograms for the different assessments used in the reported work are shown. (TIF) Figure S5 Effect of target knockdown on radiation survival in unperturbed backgrounds. A–G) Effects of target kockdown on survival of IR exposed cells. HCT116 cells transfected with target siRNA were irradiated with 2 or 5 Gy, or left untreated (control). Viable cells were quantified 5 days after IR. Data are normalized to the untreated controls. Filled triangles = target (Target), open triangles = Mock (Li). Error bars represent the variance from the mean of three biological replicates, run in triplicate each. H) Modulation of RB1 phosphoryla- tion by target knockdown. Parallel POS-LoRBPS780 analysis was used to verify siRNA performance. I) Statistical analysis: Student t-test for data shown in A–G. Note highly significant change in survival for PRKACG (***) and PRPK (**), with HK1 and p21CIP1/WAF1 strongly converging towards significance (p,0.05). K) Treatment interaction. Data were assessed for evidence of interaction between radiation and target knockdown. Values represent the degree of synergism experienced in IR exposed cells. (TIF) Figure S6 Effect of RB knockdown on radiation surviv- al. A–E) RB family knockdown affects survival of IR exposed cells. HCT116 cells transfected with oligonucleotides targeting retinoblastoma family proteins either alone, or in PLoS ONE | www.plosone.org 12 February 2012 | Volume 7 | Issue 2 | e31627