Figure 4. Human AID is sensitive to HSP90 inhibition in normal and transformed B cells.

A. Human B cells were purified from blood or tonsils, activated with IL-4 and sCD40L and 3 days later (t0) treated with 2 μM 17-AAG (+) or DMSO (−) for 24 h and analyzed by western blot using anti-AID and β-actin as loading control. The experiment was performed with 6 different donors; one representative blot of each is shown. Right, AID to actin ratio in treated cells is plotted as % of the same ratio in the corresponding untreated sample, as determined by densitometry.

B. BL cell lines EL1BL, Daudi and Ramos that constitutively express endogenous AID were treated with 8 μM 17-AAG for 24 h and AID levels analyzed by Western blot, as in (A) including densitometry of each sample (Left) or by intracellular staining and flow cytometry, with the reduction (%) in AID levels after treatment with 17-AAG indicated for each sample (Right). One representative out of 2 independent experiments is shown for each technique.

C. AID levels were analyzed by intracellular staining and flow cytometry in PBMCs from three CLL patients activated with IL-4 and sCD40L and 3 days later treated with 8 μM 17-AAG or DMSO for 24 h. The reduction (%) in AID levels after treatment with 17-AAG indicated for each sample. One representative plot from each patient is shown.