(A) BH3 profile shows increased sensitivity to Bad and Bmf peptides in EBNA3A-deleted LCLs compared to wildtype LCLs, indicative of a BCL-2 dependence. Three technical replicates with seven repeated measures each over time were averaged and plotted with SEM. Data were analyzed by paired 2-tailed t-test, *p<0.001. (B) qPCR of BCL-2, MCL-1, and BFL-1 mRNA levels in matched wild type and EBNA3A-deleted LCLs from four human donors. NS, not significant, ***p=0.002 (C) Western blot of MCL-1 in WT or △3A LCLs treated with 15 µM cycloheximide (CHX) over two hours. (D) The relative mean densitometry from panel (C) plotted over time from two biological replicates plus SEM. (E) Sub-cellular fractionation into total (T), cytoplasmic (C), or mcitochondrial (M) compartments reveals MCL-1 mislocalization in an EBNA3A-deleted LCL compared to a Wildtype LCL. Immunoblot for MCL-1, VDAC (mitochondrial localization control), and β-Actin (total lysate control). Quantified levels of MCL-1 in total and mitochondrial compartments are normalized to their respective WT levels and VDAC control. (F) Relative transcription rate of BFL-1 in WT and △3A LCLs ascertained by qPCR on pulldown of nascent mRNAs. Data is shown as the average and SEM of three biological replicates. (G) Chromatin immunoprecipitation (ChIP) was performed on extracts from WT and △3A LCLs using antibodies for total RNA Pol II (Pol II), Pol II phospho-Ser 5 (Pol II Ser 5), H3K27ac, H3K9ac, and H3K4me3. Primer pairs for Myo were used as a negative control, CXCL10 as an EBNA3A-repressed control, and miR221/222 as an EBNA3A-activated control. Primer pairs surrounding the BFL-1 TSS are shown annotated in panel (I) in their proper locations. Values represent ratio of chromatin precipitated, after correction for IgG, relative to 2.5% of input. Data are shown as the mean and standard deviation (SD). (H) Chromatin conformation capture (CCC) was performed on WT and △3A LCLs, and the BFL-1 loci were interrogated for interaction after digesting with HindIII. Relative interaction frequency to the BFL-1 TSS fragment (panel I bottom) was assayed by qPCR and normalized to the interaction frequency of the nearest neighbor (−1) fragment set at 100% relative interaction. Results are the average of two independent experiments. (I) ChIP-Seq data for EBNA2, EBNA3A, EBNA3C, NFκB (RelA), Histone H3K27ac, p300, and RNA Pol II (Pol II) from an LCL on the BCL2A1 (BFL-1) locus. (J) EBNA3A protein expression can be rescued to wild-type LCL levels in a △3A LCL using an episomal EBNA3A expression vector. (K) Rescuing EBNA3A expression in △3A LCLs restores resistance to ABT-737. Three individual clones of WT, △3A, or △3A/EBNA3A were subjected to ABT-737 treatment for three days and then analyzed for viability by FACS. Remaining viable cells on day three post treatment are normalized to the untreated (0 nM) cells. Data were analyzed by 2-way ANOVA and showed that there was a significant interaction (p<0.001) between cell type and drug treatment. NS, not significant; **p=0.01 by two-tailed t-test.