Figure 3.

53BP1 foci formation does not affect the 53BP1 signal intensity

Asynchronously growing U-2 OS cells were left untreated or treated with 2 Gy of IR and allowed to recover for 30 min. Cells were subsequently fixed with paraformaldehyde and then permeabilization with PBS-Triton 0.2% and stained for DNA content, EdU and 53BP1 signal for quantitative image-based cytometry analysis. (A) Representative images for DAPI, EdU and 53BP1 (invert LUT) staining are presented. The full size of acquired images is 2034 × 2034 pixels, an 800 × 800 pixels sector is presented for each wavelength. Scale bar: 20 μM. (B and C) Multivariate scatter plots displaying QIBC analysis representing the relationship among nuclei total DAPI intensity (X axis), mean 53BP1 intensity (Y axis) and 53BP1 foci number (color gradient) for cells left untreated or treated with 2 Gy of IR as indicated. The negative controls (in B) are the samples incubated only with the anti-Mouse Alexa-Fluor-647 secondary antibody, whereas the 53BP1 antibody staining (in C) corresponds to the experimental samples first incubated with the 53BP1 primary antibody followed by an incubation with the anti-Mouse Alexa-Fluor-647 secondary antibody. The number of analyzed nuclei (n) is indicated for each sample. (E) For the same cells shown in (D), the 53BP1 foci counts per nucleus were normalized at the single cell level to DNA content to account for increasing damage load with increasing DNA amount. (F) Box-plot representation of the number of 53BP1 foci counts per nucleus normalized to the nucleus DNA content in the indicated condition and cell-cycle phase. The bounds of the box are 25–75th percentile, center shows the median, whiskers indicate the 5–95 percentiles and the data points outside of this range are drawn as individual dots. Statistical analysis was performed with Prism (GraphPad) software. Data were analyzed with the two-tailed non-parametric Kruskal-Wallis test followed by a two-stage linear set-up procedure of Bonferroni, Kreiger and Yekutieliwas.