Hypomethylating agents induce immunogenic EBV antigens. (A,C) qRT-PCR for LMP1 and Cp promoter in cells treated with drug (decitabine or 5-azacytidine) vs vehicle control for 48 hours at the following doses listed from left to right: vehicle, 10, 25, 50, 100, 250, 500, and 1000 nM. Data are shown as fold change in treated cells compared with vehicle control. Experiments were performed in triplicate. Error bars represent SEM. (B,D) Immunoblot for viral proteins as indicated. BL cells were incubated with drug at the indicated doses for 48 hours. LCL-9001 is a latency III positive control. BC2 is a latency I control. Ramos is an EBV⁻ BL used as a negative control. Lower panel in B represents a longer exposure time for LMP1. (E-F) Immunohistochemistry for EBNA2 and LMP1 in cell blocks generated from Mutu I, Kem I, and Rael cells treated as indicated. Cells were exposed to 5-azacytidine at 4 µM, decitabine at 500 nM, or vehicle control for 48 hours. Experiments were performed in triplicate. Representative images were obtained on an Olympus BX 43 microscope (Camera, Jenoptik ProgResCF; software, ProgRes Mac Capture Pro, 2013. Original magnification ×600 with a 60/0.80 objective lens). (G-F) Image quantification using HALO (Indica labs). Error bars represent SEM. *P < .05, **P < .01, ***P < .001, ****P < .0001. 5-Aza, 5-azacytidine; DCB, decitabine; ns, not significant.