AID targeting and genomic instability in RNF8^−/− B cells undergoing CSR. (A) AID mRNA levels measured by quantitative real-time PCR performed on cDNA from B cells stimulated with LPS plus IL-4 for 48 h. The abundance of AID transcripts relative to GAPDH is shown. Error bars represent means ± SD. (B) Sµ mutations in B cells stimulated with LPS plus IL-4 and labeled with CFSE. The analysis was performed on sorted IgM⁺ cells that had undergone five divisions. The frequency of mutations per basepair sequenced and the total number of independent sequences analyzed are indicated underneath and in the center of each chart, respectively. Segment sizes in the pie charts are proportional to the number of sequences carrying the number of mutations indicated in the periphery of the charts. (C) RNF8^+/+ and RNF8^−/− B cells were stimulated with LPS and IL-4 for 72 h, IgG1-positive cells were sorted, genomic DNA was amplified by PCR, and Sμ–Sγ junctions were sequenced. The percentage of junctions with the indicated nucleotide overlap or insertion is indicated (42 RNF8^−/− and 28 RNF8^+/+ sequences were analyzed). (D) LPS plus IL-4–stimulated B cells were retrovirally transduced with p53 shRNA (see Materials and methods). At 72 h after infection, the cells were sorted and metaphase spreads were hybridized with a combination of painting probes for IgH Cα (green) and telomere-specific peptide nucleic acid probes (red), and counterstained with DAPI (blue). (left) Example of a normal metaphase spread (arrows). (right) Example of an IgH lesion (star). Bar, 5 µm. (E) The percentage of metaphases with abnormalities specifically associated with chromosome 12 is quantified. At least 100 metaphases for each group were analyzed in two independent experiments. Error bars represent means ± SD.