Fig. 1: 53BP1 forms nuclear puncta.

a MDA-MB-231 parental and 53BP1 knockout (KO) cells were treated or not with 500 nM CPT for 6 h, and protein expression was detected. Arrowhead indicates 53BP1. b Representative images of nuclear localization of 53BP1 in MDA-MB-231 cells under normal growth conditions. Single z-plane images were acquired by sequential scanning using confocal microscopy. Square: an example of puncta+ cell; Circle: a cell with three spontaneous 53BP1 foci; Arrow: a mitotic cell. Scale bar is 16  μm. c Percentage of puncta positive cells were analyzed from n = 36 individual images taken from four replicate experiments with a total n = 447 and 373 cells analyzed for parental and KO groups, respectively. Data represent mean values and standard deviation (SD). d Violin plot of 53BP1 puncta number per cell was analyzed from n = 185 and 105 parental and 53BP1 KO MDA-MB-231 cells, respectively. Data represent mean, 25th, and 75th percentiles with the whiskers extending to the minimum and maximum values. e Representative images of 53BP1 puncta and Cyclin A in MDA-MB-231 parental and 53BP1 KO cells under normal growth conditions. The cell cycle stages (G1, S/G2, and M) were determined by a combination of Cyclin A expression levels and DAPI staining patterns. Single z-plane images were acquired by sequential scanning using confocal microscopy. Scale bar is 10 μm. f Correlation between Cyclin A expression levels (expressed as mean fluorescence intensity, F.I.) and 53BP1 puncta number from n = 102 parental MDA-MB-231 cells. Each dot represents one cell. A size at or above 0.680 μm² (area) (≥25% percentile) was considered to be a 53BP1 punctum in the quantitative analyses. Unpaired two-tailed t test using Prism 9.0 was conducted for c and d with 95% confidence intervals, whereas the P-value in (f) was acquired by the Pearson Correlation Coefficient Calculator.