Extended Data Fig. 2: Recruitment of HR factors at centromeres is specific to DSB induction.
From: Activation of homologous recombination in G1 preserves centromeric integrity
a, b, Immunofluorescence confocal analysis of NIH3T3 cells expressing Cas9 + mi gRNA stained with DAPI and (a) RAD51 antibody in 568nm or (b) γ-H2AX antibody in 568nm. Images in the other two channels (488nm and 647nm) demonstrate that the RAD51 signal is very specific, and it is not a bleed through signal from different antibody staining. c, Immunofluorescence of super resolution analysis of NIH3T3 cells expressing Cas9 + mi gRNA stained with DAPI, CREST, RAD51 and BRCA1 antibodies demonstrate that RAD51 and BRCA1 were detected simultaneously on the same centromere. d, Immunofluorescence confocal analysis of NIH3T3 cells expressing dCas9 + mi gRNA stained with DAPI, 53BP1, RPA or BRCA1 antibodies. e, Immunofluorescence confocal analysis of U2OS cells expressing dCas9 + gRNA targeting the alpha satellite repeats stained with DAPI, 53BP1, RPA or BRCA1 antibodies. For confocal images, scale bars represent 5 µm. f, g, Immunofluorescence confocal analysis of (f) EdU- NIH3T3 cells expressing GFP-CENP-A and (g) thymidine treated U2OS cells for G1 and RO-3306-arrested cells for G2 treated with Neocarzinostatin (NCS, 200 ng/ml), stained with DAPI and antibodies specific for γ-H2AX, CREST and RAD51. Percentage of cells with DSBs (γ-H2AX) and RAD51 colocalizing with GFP-CENP-A or CREST, corresponding to cells with at least one centromere colocalizing with γ-H2AX and RAD51, is shown on the right as mean ± SD. For confocal images, scale bars represent 5 µm for G1 cells, 10 µm for G2 cells. For definition of p values, statistic method and sample number see Statistics and Reproducibility section.
