Fig. 3: ICRF-193 induces DSBs in repetitive sequences.

a Representative image of the pericentric region from chromosome 9, mouse mm10 genomic assembly, showing the increase in number of reads with γH2AX signal after treatment of NIH3T3 cells with ICRF-193. b–d Quantification of the increase in number of reads after γH2AX-ChIPseq analysis in NIH3T3 cells mapped to Major (b) and Minor (c) Satellite repeats, or to Long (d) and Small (e) subunits of ribosomal RNA repeats. Graphs represent average values from n = 2 biologically independent experiments. Statistical significance presented with adjusted p values by comparison with two-sided DE-Seq-2 analysis. f, g Quantification of the number of peaks identified for the indicated genomic sequences in NIH3T3 cells treated with DMSO (black) or ICRF-193 (gray) in the first (f) and second (g) biological replicates. Peak calling was performed by MACS2 and they were annotated by HOMER to general categories of genomic regions such as Exons, Introns, Promoters and transcription termination sites (TTS) and repetitive regions such as SINEs, LINEs, LTRs, Satellite and rRNA repeats. h Quantifications of tail moment in neutral comet assays performed in NIH3T3 cells. 4 biological replicates were performed for each condition, with measurements taken from 100 cells per replicate, per condition. Statistical significance presented by one-way ANOVA test. (Adjusted P values in comparison to DMSO for 0 h: p < 0.0001; 4 h: p < 0.0001). Bars represent the median value, and the error bars represent 95% CI. i Representative confocal microscopy images of exo-FISH signal (green), and DAPI (blue), with (+) or without (−) ExonucleaseIII (ExoIII) processing in NIH3T3 cells. Maximum projection of z-stacks is shown. Scale bars = 10 μm. j Percentage of NIH3T3 cells with exo-FISH signal colocalizing at heterochromatin, as shown in Fig. 3i. 2 biological replicates were performed for −ExoIII condition and 4 biological replicates for +ExoIII conditions. At least 20 cells were counted per replicate in −ExoIII and at least 50 cells were counted per replicate for the +ExoIII condition. The graph bars represent average values, and the error bars in the graphs represent the standard deviation. Statistical significance presented by two-sided student t test (P value for +ExoIII treatments: p = 0.0036). (*) p < 0.05, (**) p < 0.01, (***) p < 0.001, (****) p < 0.0001.