Fig. 3. MADD is not resolved despite intact DDR signalling in multinucleate cells.

RPE1 H2B-RFP 53BP1-GFP cells were treated with CENPEi for 24 h, washed and imaging initiated. One laser induced bleach/damage site in each nucleus was tracked. a Representative pre-bleach, bleach (at 00:00) and post-bleach images of nuclei—either normal nuclei without pre-existing 53BP1 foci, normal nuclei with pre-existing 53BP1 foci or multinucleate nuclei with 53BP1 foci. Yellow arrows indicate sites of bleaching and are highlighted in crops. Scale 15μm; insets 5μm. b Graph shows the proportion of nuclei that gain 53BP1-GFP foci at the site of laser-induced damage, for multinucleate, normal with pre-existing foci and normal nuclei without pre-existing foci. N indicates the number of cells, from across 3 independent repeats. Statistical significance was assessed using a proportions test with a 95% confidence interval. ** indicated p < 0.05. c Graph shows timing, post-bleach, of 53BP1-GFP foci arrival at the laser bleach site. d Quantification of the proportion of nuclei that cleared laser-induced 53BP1-GFP foci, for normal and multinucleate cells. Statistics was assessed using a proportions test with a 95% confidence interval. ** indicates p < 0.05. e Graph shows the timing of 53BP1-GFP foci clearance after bleach time, for foci induced at the bleach site (solid lines) and foci existing prior to laser bleach (dashed lines). N indicates the number of foci and cells analysed. f Graph shows changes in 53BP1-GFP foci intensity at laser-induced damage site in cells shown in Fig. 3a. 53BP1-GFP intensities were normalised using pre-laser damage intensity values.