DSB requirement for AID nuclear localisation.

<p>A) Western blot showing γH2AX levels in NIH/3T3 cells treated for 2 hours with 200µM etoposide or 200µg/ml bleomycin, the concentration used in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082110#B47" target="_blank">47</a>]. Histone H3 and α-tubulin are shown as loading controls, quantification of γH2AX is relative to tubulin. B) Western blot of γH2AX levels in NIH/3T3 cells after 2 hours treatment with given concentrations of etoposide and bleomycin. Controls as in A, quantification of γH2AX is relative to tubulin. C) Localisation of AID in a stable NIH/3T3 cell line expressing FLAG-AID, treated with bleomycin and etoposide at different doses for 2 hours and then allowed to recover for 5 hours. White arrows indicate cells with nuclear AID. Scale bars at bottom right of each image indicate 10µm. D) Quantification of cells shown in C with nuclear AID under different drug treatments. E) AID nuclear localisation with increasing etoposide concentration, cells treated as in C. Error bars represent ±1 s.e., * p<0.05 for Student’s <i>t</i>-test, n=3 samples per drug treatment except n=2 for 20µM etoposide and n=5 for 200µM etoposide, >100 cells were counted in each sample.