Knockdown of SMC1 or MRE11 does not influence mobility of IRIF.

<p>A) Mean square displacement (msd) of 53BP1 foci after irradiation with Pb (LET: 13500 keV/μm) is plotted against time for wt (blue line), SMC1 knockdown (red line) and MRE11 knockdown cells (green line). B) Western blots of U2OS cells 48 h after knockdown of SMC1 and MRE11 with tubulin as loading control.</p

Influence of repair-related chromatin modifying proteins on mobility of 53BP1-GFP foci in irradiated U2OS cells.

<p>A) Plot of the mean square displacement (msd) of IRIF in control cells (wt) and cells which were depleted for ATP 30 min prior to carbon ion (LET: 170 keV/μm) irradiation (n = 7). Errors represent SEM in all plots. B) Msd of IRIF in cells after knockdown of ACF1 (n = 23) and non treated controls (wt) (n = 11) after irradiation with Cr (LET: 2360 keV/μm). C) Comparison of the msd of IRIF in cells after inhibition of PARP (10 μM PJ34) and controls (wt) (n = 15). Cells were irradiated with C (LET: 170 keV/μm). D) Msd of IRIF in cells after knockdown of PARG (n = 15) and in non treated controls (wt) (n = 11). Cells were irradiated with Cr (LET: 2360 keV/μm). E) Western Blot showing the ACF1 and PARG knockdown efficiency with actin as loading control. F) U2OS cells treated with 20 mM H₂O₂ for 10 min, fixed and stained for PAR (green) and DNA (blue) show efficiency of PARP1 inhibition with 10 μM PJ34.</p

Inhibition of ATM constricts mobility of 53BP1 foci induced by heavy ion or photon irradiation.

<p>Irradiation of U2OS cells was performed by Cr (LET: 2630 keV/μm) for plots A and C and by 1 Gy X-rays for plots B and D. The mean square displacement (msd) of IRIF is plotted over time. Errors represent SEM. <b>A, B</b>) Msd plots of control (solid squares) (Cr n = 11, X-ray n = 21) and ATM inhibited cells (KU55933 open squares) (Cr n = 31, X-ray n = 11) fitted for subdiffusion (red line) and confined diffusion (blue line). <b>C,D</b>) Bar graphs of the average msd after 100 min observation time by live cell microscopy for control and ATM inhibited cells (KU55933) after irradiation with Cr <b>C</b>) and after irradiation with 1 Gy X-rays <b>D</b>). <b>E</b>) U2OS-53BP1-GFP cells irradiated with 1 Gy X-rays, fixed after 30 min and stained for pATM (red) and DNA (blue). Wt compared to cells treated with 15 μM KU55933 for 2 hours (ATMi) show efficiency of ATM kinase inhibition.</p

γH2AX signal kinetics in an extended dose range: experimental data and modelling analysis.

<p>Panel A: experimental data of flow cytometry measurements after irradiation with different X-rays doses (symbols) and corresponding application of the γH2AX kinetic model (lines); error bars show standard error of the mean for at least two independent samples. Lines show the result of the fit when input iDSB and cDSB are calculated according to Poisson distribution and assuming the standard domain size of 2 Mbp, considering also the 3D extension of the H2AX phosphorylation. Panel B: comparison between model predictions for the different doses investigated after normalization to their maximum value of the functions plotted in panel A.</p

Predicted two-dimensional H2AX phosphorylation patterns with and without the 3D spreading mechanism.

<p>Example of XY projections of hit domains only (A) and of all phosphorylated domains obtained with the random expansion algorithm (B); the exposure to 1 Gy of X-rays photon radiation was simulated. The cell nucleus is represented in light grey in the background. The colour scale indicates the multiplicity of hit domains in the column of interest.</p

Measured γH2AX dose response curve.

<p><b>γ</b>H2AX fluorescence intensity measured with flow cytometry 1 h after X-rays irradiation of AGD and CHO cells; error bars show standard error of the mean for four independent samples. Fluorescence intensity is in arbitrary units.</p

Modelling the H2AX phosphorylation in the context of Mbp-level higher-order chromatin structures.

<p>Starting from the basic assumption that chromatin loops involving about 2 Mbp of genome correspond to nuclear subunits that we define as <i>domains</i> (A), we make the hypothesis that a domain where at least one DSB is scored can be related to a phosphorylated loop, shown in green (B); importantly, no difference is expected in terms of fluorescence intensity depending on whether an iDSB or cDSB is induced, since in both cases we assume the whole loop being phosphorylated. In a further step, we consider the possibility that H2AX phosphorylation can randomly spread in a 3D fashion around the induced DSB. According to this approach, some of the domains surrounding the hit ones will produce γH2AX signal even without direct induction of DSB. This concept is schematically represented in panel C, where arrows indicate spreading of H2AX to additional domains surrounding the hit one.</p

Measured γH2AX dose response curve up to 500 Gy.

<p><b>γ</b>H2AX fluorescence intensity measured with flow cytometry 1 h after X-rays irradiation of AGD cells; the figure shows the dose response curve as extracted from the kinetics experiments (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129416#pone.0129416.g003" target="_blank">Fig 3</a>) and the dose response curve resulting from independent measurements. Error bars show standard error of the mean for three or four independent samples. Fluorescence intensity is in arbitrary units.</p

Modelling the γH2AX dose response: DSB vs hit domains.

<p>Comparison between expected numbers of induced DSB and hit domains as function of the dose; the calculations were performed assuming the standard input parameters of 30 DSB per Gy and cell nucleus and of 3000 domains (2 Mbp/domain).</p

Comparison of measured γH2AX dose response curves to model predictions including the 3D spreading mechanism for H2AX phosphorylation.

<p>Normalized γH2AX dose response curve for AGD (full squares) and CHO (open squares) cells, and corresponding model predictions of phosphorylated domains assuming random extension of H2AX phosphorylation around the hit domain; a DSB induction rate of 30 and 60 DSB/Gy was considered.</p