Induction of mitochondrial-mediated apoptosis in EBV- and KSHV-infected lymphoma cells after selective inhibition of NF-κB. (A) EBV-IBL and BC-3 cells were treated with 5 μM Bay 11 and assessed for NF-κB and octamer/DNA binding by EMSA. Nuclear proteins were extracted from cells 1 hour and 24 hours after treatment, and EMSA was performed using NF-κB or octamer-specific radiolabeled oligonucleotide probes. Cell lines demonstrated the inhibition of NF-κB throughout the 24-hour assay but no concomitant inhibition of octamer/DNA binding. (B) EBV-IBL and BC-3 cells were treated with Bay 11 and assessed for apoptosis after 24-hour treatment. For analysis, cells were labeled with FITC-conjugated Annexin-V at room temperature and were analyzed by flow cytometry. Reported results are the percentages of apoptosis cells from 4 independent experiments for untreated () and Bay 11-treated (▪) cells. Error bars represent SD. (C) EBV-IBL and BC-3 cells were cultured at 7.5 × 10⁵ cells/mL and treated with Bay 11-7082 for 24 hours. After 1, 3, 6, and 12 hours, whole cell extracts were generated and evaluated for the expression of caspases 9, 3, and 8 and PARP by Western blot analyses. Antibodies for caspase 9 and 3 recognized the full-length and cleaved caspase isoforms, which are indicated. Antibody for caspase 8 detected only the full-length or procaspase isoform, so activation was assessed by attenuated detection of the full-length protein. After initial probe, blots were stripped and reprobed with an actin control. Results shown are for BC-3 but are representative of both cell types in terms of caspase activation pattern and timing.