Evidence for EBV-infection of MECs and MEC-derived breast cancers. (A) PCR amplification of EBV genes BZLF1, BNLF1 and EBER-2 in DNA extracted from EBV-HMEC-R or GFP-HMEC-R and xenograft tumors as indicated. (B) Cell lysates from EBV-infected and control HMEC or MCF10A cells were subjected to immunoblotting with anti-LMP1 antibodies. (C) In situ hybridization of EBV-expressed RNAs (EBERs) in xenograft tumors derived from control (upper left) and EBV-infected HMECs (lower part of panel). A Burkitt's lymphoma (upper left panel) served as positive control. EBV-associated breast cancers derived from EBV-HMECs stained only infrequently positive for EBERS, with some EBERS positivity in tumor areas with cell death (left bottom). The red arrows indicate the cells with EBERS staining in the xenografts. (D) Immunoblotting of tumor lysates for CD21. (E) Heat map of viral gene expression. polyA + RNA was extracted from cell lines and xenograft tumors as indicated, and viral gene expression was measured by Q-PCR, normalized to 5 reference genes and displayied relative to the average level of 3 EBV negative samples (GFP-Tumor1, GFP-MCF-10A-R, GFP-HMEC-R). NTC, POS1 and POS10 were positive controls. Scale bars represent 100 μm.