EBV Blocks NOXA Induction Figure 5. Ionomycin induces accumulation of NOXA protein and mRNA in EBV-negative and latency I BL cells, but not in EBV- positive cells that are resistant to ionomycin-induced apoptosis. (A) Protein extracts from the cells indicated before and after exposure to 1 mg/ml ionomycin (IM) for up to 48 hours were separated by SDS-PAGE and analysed by western immunoblotting using antibodies directed against NOXA. Throughout, c-tubulin was used as a loading control. (B) EBV inhibits the accumulation of NOXA mRNA induced by ionomycin. EBV-negative and recombinant B95.8 (WT) EBV-converted BL31 and BL2 cells were exposed to 1 mg/ml ionomycin for up to 8 hours. Cells were harvested at 2-hour intervals and RNA was extracted. The extracts were analysed by quantitative RT-PCR and NOXA mRNA expression was normalized to that of UBC mRNA, which is stably expressed during ionomycin treatment/apoptosis over the time period used. doi:10.1371/journal.pone.0028506.g005 therefore important to determine whether NOXA is necessary for ionomycin-induced apoptosis in sensitive BL cells. To this end, four lentiviral vectors expressing shRNAs targeted against NOXA mRNA were used to transduce EBV-negative BL31 and BL2 cells. None of the lentiviruses alone achieved greater than a 40–50% reduction in protein level (data not shown). However, transduction of BL31 and BL2 cells with all four shNOXA lentiviruses (shNOXAx4) achieved a substantial reduction in NOXA expres- PLoS ONE | www.plosone.org 8 December 2011 | Volume 6 | Issue 12 | e28506