EBV Blocks NOXA Induction Figure 6. shRNA-mediated knockdown of NOXA protects against ionomycin-mediated apoptosis. BL31 cells were transduced with multiple (shNOXAx4) lentiviral vectors carrying shRNA directed against NOXA mRNA, or with a non-target shRNA control (shNon-target) lentivirus. (A) Western immunolotting shows the reduction in basal NOXA expression due to the shRNAs; c-tubulin was used as a loading control. (B) BL31- shNOXAx4 and BL31-shNon-target, EBV-negative BL31 and its B95.8 (WT) EBV convert were exposed to DMSO (vehicle control), or 0.5 or 1 mg/ml ionomycin (IM) for 24 hours. Cell viability was determined by CellTiter-Glo assay and expressed as a percentage of vehicle-treated controls. The mean and standard deviation of three independent experiments is shown. (C) BL2 cell lines transduced with lentiviruses as (A) above, and immunoblotted to show levels of NOXA depletion. (D) BL2 cell lines treated with ionomycin and assayed as described in (B). doi:10.1371/journal.pone.0028506.g006 sion in both cell lines. BL31 and BL2 cells were also transduced with the non-targeting shRNA-expressing lentiviruses to establish lines as negative controls (Figure 6A & C). BL31- and BL2-shNOXAx4 and shNon-target cell lines were then exposed to ionomycin and their responses compared to those of EBV- negative and WT EBV-converted BL31 and BL2. In both BL31 and BL2 cell lines, cell viability after ionomycin treatment is substantially improved as a result of NOXA knock-down, although not quite restored to the protection offered by EBV infection (Figures 6B and D). Similarly, PARP cleavage is reduced or prevented in the absence of NOXA, in both BL31 and in BL2 cells (Figure S5). These observations are consistent with either an incomplete ‘knockdown’ of NOXA, or with the possibility that NOXA represents a partial component of the apoptotic stimulus caused by ionomycin, but either way, confirm that NOXA makes a major contribution to the apoptosis induced in these cells by ionomycin. PLoS ONE | www.plosone.org 9 December 2011 | Volume 6 | Issue 12 | e28506