a, Upper: Diagram of IgH FISH probes. An intact IgH shows co-localized red and green signals while a broken locus appears as split red and green signals. Middle: Example of metaphase FISH showing IgH breaks. Lower: Quantification of IgH abnormalities in day4 αCD40/IL4-activated control (n=6), CX^c/− (n=9), CX^c/−A^−/− (n=5) and CX^c/−RAG^c (n=8) splenic B cells (details in Suppl. Table 1). b, Upper: Diagram of Igλ FISH probes. Intact Igλ shows co-localized green and red signals, Igλ breaks appear as split green and red signals, either free or in translocations. Middle: Examples of metaphase FISH showing Igλ breaks (left) and an Igλ break and dicentric translocation (right). Lower: Quantification of Igλ abnormalities in day4 αCD40/IL4-activated control (n=11), CX^c/− (n=11), CX^c/−A^−/− (n=3), CX^c/−RAG2^c (n=8) splenic B cells (details in Suppl. Table 3). c, Upper: Diagram of Igκ FISH probes. Igκ breaks are scored similarly as Igλ breaks. Middle: Examples of metaphase FISH showing an Igκ break (left) and Igκ break and translocations (right), involving both centromeric and telomeric portions of chromosome 6. Lower: Quantification of Igκ abnormalities in day4 αCD40/IL4-activated control (n=10), CX^c/− (n=11), CX^c/−A^−/− (n=3), CX^c/−RAG^c (n=7) splenic B cells (details in Suppl. Table 4). d, Upper: Diagram of Igλ 3D interphase FISH Probes. Middle: Representative 3D interphase FISH showing intact Igλ (co-localization of green and red signals) and Igλ breaks (split green and red signals) (details in Suppl. Fig. 4). Lower: Quantification of Igλ abnormalities by 3D interphase FISH on day 0 (n=3) or day 4 (n=3) αCD40/IL4-activated splenic B cells. We could not do similar assays for Igκ due to the large size of this locus (greater than 3Mb). In all panels, data are presented as mean ± s.e.m. Statistical analyses were calculated by a Student's t-Test with two-tailed distribution.

a, Top: Diagram showing 3′IgH (green) and 3′Igλ (red) probes used for 3D interphase FISH. Bottom: Representative co-localization of IgH/Igλ in day 0 control and CX^c/− B cell interphase nuclei. b, Schematic map of Igλ, C2 and K10 BAC probes on chromosome 16. c, Quantification of co-localization of IgH-Igλ, IgH-C2, or IgH-K10 loci in nuclei of day 0 control and CX^c/− splenic B cells and in nuclei of thymocytes (details in Suppl. Tables 9 and 11). d, Quantification of co-localization of IgH-Igλ, IgH-C2, or IgH-K10 loci in day3.5-activated control or CX^c/− peripheral B cells (details in Suppl. Table 10). At least three mice were analyzed per data set; data are presented as mean ± s.e.m.

a, Top left: Diagram showing 3′Igλ probe (green) on chromosome 16 and 3′IgH probe (red) on chromosome 12. Bottom left: Representative Igλ/IgH translocation showing green and red signals juxtaposed on a dicentric chromosome (yellow arrow). Right: Quantification of IgH/Igλ translocations in day4 αCD40/IL4-activated control (n=2) or CX^c/− (n=4) B cells analyzed by metaphase FISH (details in Suppl. Table 8). Data are presented as mean ± std. b, PCR-isolated Igh/Igλ translocation junctions from day4 activated CX^c/− B cells (n=3) (primers indicated by horizontal black arrows). Junctional sequences are shown in Suppl. Fig. 11. A vertical green arrow indicates breakpoints. For a given translocation, the same number is used to indicate the corresponding IgH and Igλ breakpoints, with the IgH breakpoint denoted by a (') symbol.

a, Frequency of IgH/c-myc translocations from day4 αCD40/IL4-activated wt (n=4) and CX^c/− (n=4) splenic B cells, or B cells harboring c-myc^25ISceI/wt (n=3) or c-myc^wt/wt (n=1) infected with either control or ISceI-expressing retrovirus (details in Suppl. Fig. 13 and 15). b, Top: Schematic showing c-myc (red) probe on chromosome 15 and 3′IgH (green) probe on chromosome 12. Bottom: Representative images of IgH/c-myc co-localization in day 0 control and CX^c/− B cell interphase nuclei. c, Quantification of IgH/c-myc association by 3D interphase FISH in control and CX^c/− splenic B cells (n=3), and ES cells (n=3). Cells were analyzed at the indicated time points before or after stimulation. d, Schematic showing the wt c-myc allele (c-myc^wt) and the modified c-myc allele containing 25 ISceI sites (c-myc^25ISceI). e, Top left: Diagram of c-myc FISH probes. Bottom left: Representative c-myc abnormalities in αCD40/IL-4-activated c-myc^25ISceI/wt B cells infected with IsceI-expressing retrovirus, appearing as green and red signals on separate chromosome fragments (white arrows). Bottom right: Quantification of c-myc breaks by metaphase FISH on day4 αCD40/IL4-activated B cells harboring either c-myc^25ISceI/wt (n=4) or c-myc^wt/wt (n=1) alleles after infection with control or ISceI-expressing retrovirus. Data are presented as mean ± std (details in Suppl. Table 16). High titer retrovirus infection appears to inhibit end joining allowing break visualization (see online methods).