Fig. 8

Nuclear exclusion is necessary to enforce AID licensing. a (Left) Representative confocal microscopic images of AID-GFP and AIDΔE5-GFP fusions in CH12 B cells. (Right) Nuclear GFP signal was measured as the overall GFP signal overlapping with a nuclear mask, generated using Dapi signal. Cells expressing each construct were fixed and imaged with identical settings in parallel. Individual cell values (dots) are plotted relative to the median of AID-GFP, from one experiment. Control shows untransfected cells signals. Differences were evaluated by unpaired, two tailed t-test. b Relative mutagenic activity of AIDΔE5 variants in E. coli, measured as the frequency of rifampicin-resistant (Rif-R) colony-forming units (cfu). Means (bars) of medians (dots) from 2 independent experiments (5 cultures/experiment), normalized to AID. c Effect of AID or AIDΔE5 variants-ires-GFP on the competitive growth of transduced AID-deficient CH12 B cells. Means GFP⁺/GFP⁻ ratio ± s.e.m. over time from two independent experiments, each normalized to maximal value. d (Left) Representative confocal microscopic images of GFP and γH2AX immunofluorescence with DNA staining (Dapi) in Aicda^−/− mouse B cells activated with LPS and IL-4 complemented with AID variants-ires-GFP vectors. (Right) The proportion of cells with ≥5 γH2AX foci per nucleus is plotted from 1 experiment, with n cells counted. a, d Magnification 630×. Scale bar, 10 μm. e SHM capacity of AIDΔE5 variants-ires-GFP measured by IgM-loss over time in complemented DT40 Aicda^−/− ΔΨVλ B cells. One of the two independent experiments is shown. f Number and frequency (mutations per base pair) of mutations scored at the IgV of DT40 Aicda^−/− ΔΨVλ B cells expressing AIDΔE5 variants obtained from GFP+ cells sorted at day 3 post-transduction (see e) from one experiment. Mutation load pie charts, with slices representing proportion of sequences with the indicated number of mutations and total sequences analysed indicated in the centre. g (Left) Mutation profiles and (right) mutation load, as in f scored at the Sμ region of Aicda^−/−Ung^−/− mouse B cells transduced with AIDΔE5 variants. h WB of cell extracts from reconstituted mouse B cells probed with antibodies recognizing GFP and the N-terminus of AID. For gel source data, see supplementary Fig. 7. i Model for AID targeting (see Discussion)