EBV Blocks NOXA Induction Figure 1. Latent EBV inhibits ionomycin-induced apoptosis in a variety of cell backgrounds. (A) BL41 and its EBV-positive counterparts, BL41/B95.8 and BL41/P3HR1, and two Wp-restricted BL Oku-BL and Sal-BL, (B) BL2 and its EBV-positive convert established with a recombinant EBNA2-knockout virus (BL2+E2KO), (C) Mutu III (expressing all the latency-associated proteins) and Mutu I (expressing only EBNA1) and (D) two LCLs were all treated with 1 mg/ml ionomycin (IM) for up to 48 hours. Viable cells (assessed by trypan blue exclusion) were counted. Cell survival is expressed as a percentage relative to the starting population and the mean and standard deviation of viable cell counts from three independent experiments are shown. doi:10.1371/journal.pone.0028506.g001 latency (EBNA1 and the EBERs) is insufficient to protect against ionomycin-induced apoptosis. Since the experiments described so far utilized BL-derived cells, we determined whether two lymphoblastoid cell lines, LCL-BF and LCL-CH, established by infection of primary B cells with the B95-8 strain of EBV [30] show the same resistance to ionomycin as type III BL cells. Viable cell counts for both these LCLs showed that at least 90% of cells survived after 48 hours of exposure to ionomycin (Figure 1D). PARP is also unaltered by treatment in both LCLs, confirming that these LCLs failed to undergo apoptosis even after exposure to ionomycin for 48 hours (Figure S1). These results are in marked contrast to the response of LCLs to genotoxic drugs such as cisplatin and nocodazole, since EBV cannot protect against genotoxin-induced, p53-mediated apoptosis in LCLs [23]. EBNA3A, EBNA3B and EBNA3C are not involved in protection against ionomycin-induced apoptosis Expression of both EBNA3A and EBNA3C is necessary to inhibit genotoxin-induced apoptosis in a p53-mutant background [21,22], we therefore asked whether expression of EBNA3A, EBNA3B and EBNA3C is necessary for resistance to ionomycin in PLoS ONE | www.plosone.org 3 December 2011 | Volume 6 | Issue 12 | e28506