RB1 architecture and study design.

<p><b>A.</b> Schematic of RB1 domain structure. RB1 NH2-terminal domain (RB-N, light blue), RB1 pocket-domain (RB-P, raspberry), the position of the twin cyclin folds which form the core of each domain is indicated, RB1 C-terminal region (RB-C, yellow)<b>. B.</b> RB1 constructs used in this study indicating the range of amino-acids covered. In the MBP-RB-NP and MBP-ddRB-NP constructs maltose binding protein (MBP, green) is coupled to the N-terminus of the RB construct, while in ddRB-NP-MBP it is coupled to the C-terminus. In the ddRB-NP, MBP-ddRB-NP and ddRB-NP-MBP constructs two interstitial regions were deleted, corresponding to residues 250–269, the arginine-rich linker (R-linker) of the RB-N domain, and residues 579–643, corresponding to the pocket linker connecting RB-P domain pocket lobes (P-linker). The positions of cyclin-dependent kinase consensus sites in RB-NP are indicated, with sites retained in the ddRB-NP, MBP-ddRB-NP and ddRB-NP-MBP constructs bold and starred. <b>C.</b> Atomic models of the RB-N and RB-P domains, shown in ribbon representations. RB-N left, RB-P right. Cyclin-fold helixes are coloured, RB-N A-fold in cyan, RB-N B-fold in light blue, RB-P A-fold in dark salmon, RB-P B-fold in pink, other helixes and visible loops are shown as grey.</p

Binding surfaces positioning in the active and inactive structure, and predicted molecular movement to yield inactive RB1. A., B.

<p>Relative orientation of the functional surfaces in the model of active, nonphosphorylated (A) and inactive, phosphorylated (B) RB1. Cartoon representation of Rb-NP with overlaid transparent surface with RB-N in light blue, RB-P in light-pink. The residues involved in docking LXCXE are shown in yellow, those forming the FXXXV motif are shown in purple and those for EXXXDLFD in cyan. The residues 346–355 which form a helix in unmodified RB-N but are disordered in inactive RB-NP are represented in dark grey <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058463#pone.0058463-Hassler1" target="_blank">[17]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058463#pone.0058463-Burke1" target="_blank">[21]</a>, amino acid groups involved in RB-N:P interphase interaction in the inactive conformation in red ([RB-N K136, D139, T140, T142, D145], [RB-P Q736, E737, K740, K729]) and orange [(RB-N L161, K164, L206-E209, L211-I213, F216, E282, E287, N290, N295] [RB-P Q736, E737, K740, K729]). <b>C., D.</b> Cartoon representation of active, nonphosphorylated, (C) and inactive, phosphorylated (D) RB1. RB-N B-fold is coloured in green and RB-P B-fold in purple (this different colour scheme has not been used elsewhere in the paper and is only used here for clarity). The residues 346–355 which are structured in unmodified RB-N and unstructured in inactive RB-NP are represented in dark grey. <b>E.</b> Predicted molecular movement yielding conformational RB1 inactivation. Note surfaces involved in binding LXCXE motif proteins in RB-P (salmon/pink) and the homologous surface involved in FXXXV binding in RB-N (cyan/blue) are collinear in the active (left) but not inactive form (right).</p

Characterisation of RB1 derivatives by small-angle X-ray scattering.

<p><b>A</b>. Experimental and calculated scattering patterns of ddRB<b>-</b>NP (1), MBP-ddRB<b>-</b>NP (2), ddRB<b>-</b>NP-MBP (3). Experimental SAXS data as black dots with black error bars. Lines (red) represent the fits from <i>ab initio</i> models shown in <b>C</b> (ddRB-NP), <b>D</b> (MBP-ddRB-NP) and <b>E</b> (ddRB-NP-MBP). The logarithm of the scattering intensity is plotted as a function of momentum transfer, s = 4πsin(θ/2)/λ where θ is the scattering angle and λ is the wavelength of the X-rays (1.5 Å). <b>B.</b> Distance distribution functions for ddRB-NP, MBP-ddRB-NP and ddRB-NP-MBP. <b>C.</b> Averaged <i>ab initio</i> models for ddRB-NP obtained using DAMMIN (grey semi-transparent spheres) and MONSA (RB-N blue spheres, RB-P red spheres) superimposed. The models are shown in two different views rotated by 90°. <b>D., E. </b><i>Ab initio</i> models of MBP-ddRB-NP (<b>D</b>) and ddRB-NP-MBP (<b>E</b>) obtained by MONSA. MBP is shown as green, ddRB-NP as grey spheres. The models are viewed as in <b>C</b>. <b>F.</b> Radius of gyration (<i>R_g</i>) distribution obtained by EOM for ddRB-NP. Distributions correspond to a random pool of 10.000 generated structures (blue) and the EOM optimized ensemble (red).</p

Single particle analysis of electron microscope images of MBP-ddRB-NP. A.-F.

<p>3D reconstruction of unmodified MBP-ddRB-NP. <b>A.</b>, <b>B</b>. Single particle reconstruction for unmodified MBP-ddRB-NP. Calculated density map of MBP-ddRB-NP, shown as surface representations in grey related by a 90^o rotation. <b>C.</b> 3D reconstruction in mesh representation oriented as in <b>B</b> with the docked structures of the RB-N and RB-P domains (PDB codes 2QDJ and 3POM) shown as cartoons colour-coded as follows: RB-N domain lobe A -cyan, lobe B -light blue; RB-P domain lobe A -dark salmon and lobe B – pink. <b>D., E.</b> Segmented densities shown as solid surface representation with overlaid surface representation of the unmodified RB-NP 3D reconstruction in mesh. The density attributed to the MBP tag is shown in light green, that attributed to RB-N in light blue and to RB-P in light pink. <b>F.</b> Docked structures of the RB-N and RB-P domains (PDB codes 2QDJ and 3POM) without density mesh, shown as cartoons and colour-coded as in C. <b>G.–L.</b> 3D reconstruction of phosphorylated MBP-ddRB-NP. <b>G., H</b>. 3D reconstruction shown as a grey surface in two orthogonal views. <b>I.</b> 3D reconstruction in mesh representation oriented as in <b>H</b> with the docked structures of inactive RB-NP (PDB code 4ELJ) shown as cartoons colour-coded as follows:-. RB-N domain lobe A -cyan, lobe B -light blue; RB-P domain lobe A -dark salmon and lobe B – pink. <b>J., K.</b> Segmented densities shown as solid surface representation with overlaid surface representation of the 3D reconstruction in mesh<b>.</b> Same colour coding as in <b>D</b> and <b>E. L.</b> Docked structures of inactive RB-NP (PDB code 4ELJ) without density mesh, shown as cartoons colour-coded as in I.</p

Data consolidation.

<p><b>A</b>) <b>Data clustering analysis.</b> Unsupervised clustering based on numerical observations. <b>B</b>) <b>Signalling model.</b> Hits split into groups according to their role in the accumulation of the TP53 target p21^CIP1/WAF1. Canonical double strand signalling components (ATM, ATR, CHK1/2) affecting the TP53/p21^CIP1/WAF1 axes in conjunction with S/G2 checkpoint activation do not affect G1 checkpoint function.</p

siRNA screen for gene products involved in IR-associated RB1 activation.

<p><b>A</b>) <b>Loss of RB1 phosporylation in IR exposed cells.</b> 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>B</b>) <b>Loss of RB1 phosphorylation is TP53-dependent.</b> TP53 positive (+/+) and isogenic TP53 null (−/−) 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 p21^CIP1/WAF1 were established by immunoblotting. <b>C</b>) <b>IR affects RB1 phosphorylation at multiple sites.</b> 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. <b>D</b>) <b>siRNA screening strategy.</b> 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. <b>E</b>) <b>Radio-resistant RB1 phosphorylation in cells with siRNA-mediated TP53-signalling knockdown.</b> Assay set up was as described in D, siRNA pools for TP53, p21^CIP1/WAF1 or a non-targeting oligonucleotide (nt) were used for transfection. Error bars relate to variance in POS-LoRBPS780 values from triplicate wells. <b>F</b>) <b>Primary screen outcome.</b> 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.</p

Effect of target knock down on G1 checkpoint activation.

<p><b>A</b>) <b>Effect of target knockdown on relative G1 positivity.</b> 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>B</b>) <b>Modulation of RB1 phosphorylation by target knockdown.</b> POS-LoRBPS780 analysis performed in parallel to A). Data points represent the means of triplicate technical replicates. <b>C</b>) <b>Statistical analysis.</b> Paired t-tests for data shown in A. <b>D</b>) <b>Treatment interaction test.</b> Data were assessed for evidence of a interaction between radiation and target knockdown. Values indicate the degree of antagonism experienced in IR exposed cells.</p

Effect of target knockdown on radiation survival.

<p><b>A–I</b>) <b>Target knockdown affects survival of IR exposed cells.</b> HCT116 cells transfected with target siRNA alone or in combination with siRNA targeting CHK1. Cells were Mock-irradiated or irradiated with 2 Gy or 5 Gy and viable cells quantified 5 days later. Data plotted are normalized to the respective untreated controls. Filled squares = combined target and CHK1 knockdown (Target/CHK1), open squares = CHK1 only (Li/CHK1), filled triangles = Target only (Target/NT), open triangles = Mock (Li/NT). Error bars represent the variance from the mean from three technical replicates. <b>K</b>) <b>Modulation of RB1 phosphorylation by target knockdown.</b> Parallel POS-LoRBPS780 analysis was used to verify siRNA performance. <b>L</b>) <b>Statistical analysis.</b> Student t-test for cell viability data shown in A–I, (Li/NT vs Targ/NT) probing for a significant effect of target knockdown in unperturbed cells, (Li/CHK1 vs Targ/CHK1) probing for a significant effect of target knockdown in CHK1-perturbed cells, Target/NT vs Target/CHK1 probing for a significant effect of CHK1 knockdown in target-perturbed cells. <b>M, N</b>) <b>Treatment interaction.</b> Assessment for evidence of interaction between radiation and target knockdown. Values represent the degree of net synergism between target knockdown and IR in NT (M) or CHK1-perturbed (N) cell background.</p

Hit gene-ontology and pathway associations.

<p><b>A</b>) <b>Pathway representation within hit pool.</b> Hits were analysed for pathway association using the DAVID functional annotation tool (<a href="http://david.abcc.ncifcrf.gov/" target="_blank">http://david.abcc.ncifcrf.gov/</a>). <b>B</b>) <b>Enrichment for gene ontology.</b> Pathway association was analysed for hits and input using DAVID. Pathway representation within hits is plotted against that for input targets. <b>C</b>) <b>Hit validation.</b> Hits were assessed using individual oligonucleotides represented within the pool. The number of active oligonucleotides and level of response is indicated. Hit classification was as for the screen.</p

Effect of target knockdown on IR-mediated p21^CIP1/WAF1 induction.

<p><b>A</b>) <b>Effect of target knockdown on p21^CIP1/WAF1 positivity.</b> HCT116 cells transfected with siRNA as indicated were irradiated (IR) or left untreated (control). Cells were assessed for p21^CIP1/WAF1 positivity 16 hrs post IR. The percentage of cells with p21^CIP1/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>B</b>) <b>Modulation of RB1 phosphorylation by target knockdown.</b> 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. <b>C</b>) <b>Statistical analysis.</b> Paired t-tests results for data shown in A. <b>D</b>) <b>Treatment interaction test.</b> Targets that yielded significant impairment of p21^CIP1/WAF1 positivity were tested for evidence of interaction between radiation and target knockdown. Values indicate the degree of antagonism experienced in IR exposed cells.</p