Effect of BCR stimulation on B cell infection with LMP2 KOs.

1×10⁶ purified B cells were labeled with Violet Tracer (Invitrogen) and infected with recombinant wt and LMP2 KO EBV at MOI 1. 10 ug/ml sIg (Jackson Immunoresearch) was added to each sample for BCR stimulation. B cells were harvested at 4, 7 and 14 days post-infection and stained for Annexin V. During fixation step, 10 μl counting beads (CountBright Absolute Counting Beads, Invitrogen) were added to each sample. During acquisition, the event gate was set to 5000 beads, which normalized the acquisition volume between samples and allowed for accurate, absolute counts of proliferating B cells. (A) Proliferation and (B) apoptosis data points are an average of three independent experiments ± SEM. (C) (D) RNA harvested at specific time points (12, 24, 48,72, 96, 120 and 168 hours post-infection/BCR stimulation) for analysis using Real Time PCR. (C) One latent gene (LMP1) and (D) one lytic gene (Zebra) analyzed for each time point to determine lytic induction. Statistical significance determined using Two-way ANOVA and Bonferroni Post-test (Figure 7C & 7D – each data point for LMP2 knockout viruses was compared to wild-type infection to determine statistical significance). P-value (*) <0.05, p-value (***) <0.001 and p-value (****) <0.0001.