Extended Data Fig. 7: Assessment of genomic instability associated with lack of HR at centromeric DSBs in G1.
From: Activation of homologous recombination in G1 preserves centromeric integrity
a, Immunofluorescence confocal analysis of NIH3T3 cells treated with RAD51 inhibitor (RAD51i) and irradiation (IR) and stained with DAPI and antibodies specific for γ-H2AX and RAD51; RAD51i was washed out or not after IR. b, Quantification of the number of translocations per 50 metaphase spreads in cells expressing Cas9 + mi gRNA and depleted of RAD51 (siRAD51), or of RAD51 and RAD52 (siRAD51+siRAD52) and compared to siSCR control. c, Quantification of RAD51 and RAD52 mRNA by RT-qPCR in NIH3T3 cells expressing siRAD51 or siRAD52, normalized to GAPDH and relative to siSCR. d, Quantification of the number of chromosomes with broken centromeres (lacking or with free centromeres) for 50 metaphase spreads in cells expressing Cas9 + mi gRNA and co-expressing siRAD51, or siRAD51+siRAD52 compared to siSCR. e, Quantification of fold enrichment of centromeric DNA after qPCR analysis in U2OS cells expressing Cas9 + gRNA targeting the alpha satellite repeats and siRAD51 alone or together with siRAD52 relative to siSCR control. f, Quantification of RAD52 mRNA by RT-qPCR in siRAD52 U2OS cells, normalized to GAPDH and relative to siSCR (left panel). Western blot analysis of RAD51 in siRAD51 U2OS cells compared to siSCR (right panel). g, Immunofluorescence confocal analysis of NIH3T3 cells expressing Cas9 + mi gRNA and RAD52-GFP, depleted of RAD51 (siRAD51) compared to siSCR and stained with DAPI and antibodies specific for γ-H2AX and RAD51. h, Quantification of the number of translocations in WT and PolQ−/− MEFs expressing Cas9 + mi gRNA and treated with RAD51i or DMSO as a control. For confocal images, scale bars represent 5 µm. For definition of p values, statistic method and sample number see Statistics and Reproducibility section.
