Figure 4.

decreased 53BP1 foci formation by 53BP1 siRNA

Asynchronously growing U-2 OS cells were left untransfected or transfected with control siRNA (siCTL) or 53BP1 siRNA (si53BP1) Individual siRNA transfections were performed for 72 h with siRNA from Dharmacon using Lipofectamine RNAiMAX. The siRNAs used were the Non-targeting Pool (siCTL); ON-TARGET plus Smart pool Human TP53BP1 siRNA (si53BP1). 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) 53BP1 level assessed by immunoblotting. Individual siRNA transfections were performed for 72 h with the indicated siRNA. Cells were lysed in 8 M urea, 50 mM Tris HCl, pH 7.5 and 150 mM β-mercaptoethanol, sonicated and heated at 90°C for 10 min. Samples were subjected to electrophoresis in NuPAGE Novex 4–12% Bis-Tris pre-cast gels (Thermo Fisher Scientific, Catalog # NW04120BOX), proteins were subjected to western transfer onto the nitrocellulose membrane with the NuPAGE Transfer Buffer. Primary antibodies for immunoblotting were used the following concentrations: mouse anti-53BP1 antibody (1:1000), mouse anti-α-Tubulin antibody (1:1000) and secondary horseradish peroxidase conjugated goat anti-mouse IgG antibody (1:10000). (C) Multivariate scatter plots of QIBC analysis presenting the relationship among nuclei total DAPI intensity (X-axis) and 53BP1 foci counts per nucleus normalized at the single cell level to DNA content (Y-axis). The untransfected sample was incubated only with the anti-Mouse Alexa-Fluor-647 secondary antibody, whereas the siCTL and the si53BP1 experimental samples were first incubated with the 53BP1 primary antibody followed by an incubation with the anti-Mouse Alexa-Fluor-647 secondary antibody. Number of analyzed nuclei (n) is indicated for each sample. (D) Box-plot representation of the number of 53BP1 foci counts per nucleus normalized to nucleus DNA content for 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.