a. DIvA cells were transfected with the indicated siRNA, treated with 4OHT (4h) and subjected to γH2AX staining. Image acquisition was performed using a high throughput microscope. Average foci size (x axis) and number of foci (y axis) were determined in each cells and plotted against each other. The percent of cluster positive cells relative to the entire population were calculated as described in Fig.6. Left panels show a representative experiment and right panels show the mean and s.e.m of cluster positive cells in independent experiments (RNF8, XRCC4, n=3; Sun1 n=5; 53BP1 n=4). ns, non-significant (paired t-test). b. Clustering index upon depletion of MRE11, NBS1, 53BP1, XRCC4, or RNF8 (top panel) or SUN1, SUN2 and FMN2 (bottom panel) by siRNA was measured as described Fig. S5b. Clustering index in cells transfected with control siRNA is set to 1. The mean and s.e.m of 4 independent experiment is shown. * p<0.05, ** p<0.01, *** p<0.005 (one sample t-test). c. Clustering was analyzed by high throughput microscopy in 4OHT treated DIvA untreated (NT) or pre-treated with DRB (100μM). A representative experiment is shown. d. Quantification of cluster positive cells is shown for two independent experiments upon DRB treatment at 100μM (top panels) or as the mean and s.e.m for 3 independent experiments upon DRB treatment at 20μM (bottom panel), p=0.0003 (paired t-test).