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. 2020 Nov 13;11:5776. doi: 10.1038/s41467-020-19504-3

Fig. 2. DSB induction initiates a nuclear wide relocalization of 53BP1 dimers to DSB sites.

Fig. 2

a, b Co-localization of eGFP-53BP1 with γH2AX immunofluorescence confirms 53BP1 foci correlate with DNA DSBs in live DIvA cells (a) and enrich these nuclear locations (b). c Intensity images of NB FFS measurements acquired in a single DIvA cell expressing eGFP-53BP1 before and at different time points after 4OHT induction of DNA DSBs. d Brightness maps of the NB data acquisitions presented in c pseudo-colored according to the brightness windows defined in Fig. 1i. Corresponding intensity vs. brightness scatter plots are presented in Supplementary Fig. 3a and additional NB FFS data acquired at – 10 min and 60 min 4OHT treatment in Supplementary Fig. 3b, c. e Intensity mask highlighting the nucleoplasm (Nuc) (left panel) and quantification of the fractional contribution of 53BP1 monomer (teal), dimer (green) and oligomer (red) within this region of interest before and at different time points after 4OHT induction of DNA DSBs (right panel). f NB quantification of the fraction of 53BP1 dimer present in the nucleoplasm before and 60 min after 4OHT induction of DNA DSBs (N = 10 cells, two biological replicates). g Intensity mask highlighting the foci (left panel) and quantification of the fractional contribution of 53BP1 monomer (teal), dimer (green) and oligomer (red) within this region of interest before and at different time points after 4OHT induction of DNA DSBs (right panel). h NB quantification of the fraction of the nucleus occupied by 53BP1 foci before and 60 min after 4OHT induction of DNA DSBs (N = 10 cells, two biological replicates). i, j. Homo-FRET quantification of eGFP-53BP1 dimer and or oligomer formation in the nucleoplasm of DIvA cells before vs. after 4OHT treatment (i) and in the nucleoplasm vs. foci of DIvA cells after 4OHT treatment (j) (N = 8 cells, two biological replicates). Box and whisker plots in f, h–j show the minimum, maximum, sample median, and first vs. third quartiles. *P < 0.05 (unpaired t-test). Scale bars, 5 μm.