Effect of DC mediated AID induction on genomic instability. (A-B) DC-mediated induction of genomic instability in human MM. (A) MM cells (U266, OPM2) isolated from cocultures with Mo-DCs were analyzed for the presence of γ-H2AX foci by immunofluorescence microscopy indicative of the formation of DNA DSBs. Top panel shows 2 representative nuclei with γ-H2AX foci. Bottom panel shows quantitation of nuclei with γ-H2AX foci in the cytospins. (HPF indicates high-power fields). (B) Lysates from U266 and OPM2 cells were also analyzed by Western blot analysis for the expression of p-H2AX, as a marker for genomic damage. (C) DC-mediated induction of genomic instability is AID dependent. MM cells were electroporated with AID or nontargeting (NT) siRNA (as a control) to inhibit AID before coculture with DCs. Tumor cells were analyzed by immunofluorescence microscopy for the detection of γ-H2AX foci. (D) Role of RANK/RANKL interactions in DC-mediated induction of genomic instability. MM cells (U266) were cocultured with Mo-DCs in the presence of IgG or OPG (0.5 μg/mL) to inhibit RANK/RANKL interactions or human IgG as a control. The formation of γ-H2AX foci was monitored by immunofluorescence microscopy as in panel A. Data are represented as percentage change in the number of cells exhibiting γ-H2AX foci relative to control.