Fig 1.
Kinetics of NBS1 focus frequency in control and irradiated cells.
Dots represent the experimental data points and the shaded corridors represent the standard errors of the mean (SEM). Time 0 denotes the start of irradiation. Images were acquired every minute for a period of 300 min. The expected frequencies calculated on the basis of the additivity assumption are displayed as a black dashed line. A statistically significant difference (one-way ANOVA) was obtained for all pairs of treatments, except for foci formed after irradiation with γ versus α → γ.
Table 1.
Observed frequencies of NBS1 foci per cell nucleus in control cells, cells exposed to α and γ radiation alone, to α → γ and γ → α, as well as predicted values based on assumption of additivity.
The initial number of foci per nucleus is reported as the mean number of foci recorded in the 16th minute after start of irradiation (16th minute is the first minute of image series recorded for combined irradiations). The final number of foci per nucleus denotes the mean number of foci recorded in the 310th minute, which is the last minute of image acquisition of cells treatment with γ.
Fig 2.
Kinetics of radiation-induced focus area described by polynomial fits (panel A). Corridors represent the uncertainties of fits. Data points are omitted for the sake of clarity but complete graphs can be found in supporting information material (Fig B1 in S2 Appendix). Area distributions of foci detected during 0–99 min are shown in panel B and during 100–300 min in panel C.
Table 2.
Fit parameters of normal distributions describing the focus area histograms.
Uncertainties indicate standard deviations. Differences between the early and late interval are considered statistically significant at p <0.05.
Fig 3.
Polynomial functions fitted to average total focus intensities per cell nucleus during 300 min post time 0.
Corridors represent uncertainties of fit. A graph including the data points can be found in the supporting information (Fig C1 in S3 Appendix).
Fig 4.
Relative focus intensities per cell nucleus during 300 min post time 0.
Relative values were obtained by dividing the total focus intensities (shown in Fig 3) by the average background intensity (the mean intensity of nuclear area without foci from which the mean total focus intensity was subtracted). Corridors represent standard errors of the mean.
Fig 5.
Exemplary images of nuclei recorded at given selected time points with foci induced by alpha radiation.
Selected time-frames are shown. Top row: raw images of nuclei with foci. Bottom raw: same images as in the top row but with foci identified by the software. The image montage was done using the ImageJ 1.53r, version (https://imagej.nih.gov). Three repair foci and their positions in the 5th, 15th, and 25th minute of observation are marked in top row images to demonstrate the upward movement of focus nr 2. Exemplary films demonstrating focus kinetics after γ → α and α → γ can be found the link: https://github.com/anna-irda/nbs1/wiki.
Fig 6.
Mean square displacement (MSD) of RIF induced by alpha radiation, gamma radiation, and mixed beams of both sequences.
Solid lines represent fits to data points. Corridors represent standard errors of the mean.
Table 3.
Mean square displacement analysis with parameters for fitting the function of the sub-diffusion model.
Radiation types are ranked according to the product of Dc and rc (divided by 1000 for clarity). All parameters describing the focus movement for γ → α are significantly higher from the others (p < 0.0001 with one-tailed t-test).