Figure 2: Effect of vtRNA levels on EBV infection and apoptosis.

(a) Northern blot analysis reveals the amount of ectopically expressed vtRNA1-1, 1-2 and 1-3 in BL41 cells. The 5.8S rRNA serves as internal loading control. See also Supplementary Fig. 5. (b) Fluorescence-activated cell sorting (FACS) analysis to investigate the effect of infecting the BL41 cell line with the recombinant EBV virus 2089 (ref. 32) in the absence (−) or presence of ectopically expressed vtRNA1-1 or vtRNA1-2. The data shown represent the mean and standard deviation of 11 independent experiments. The GFP-positive (pos.) Raji cells after EBV infection (infection efficiency typically 30%) served as positive infection control and were taken as 1.0. (c) The same cells as in b were labelled with annexin V PE and apoptotic cells were recorded on FL-2. (d) BL41 cells without (−) or with vtRNA1-1, 1-2 or 1-3 expression were treated with staurosporine (Stau) or etoposide (Eto) and the amount of apoptotic cells was determined by FACS analysis after an annexin stain. The data originates from five independent experiments. The number of apoptotic cells in the untreated control was subtracted in each individual experiment from the staurosporine- or etoposide-treated ones. The numbers of apoptotic cells in the untreated controls were <12%. (e) BL41 cells without (−) or with vtRNA1-1 expression were treated with anti-Fas antibody and the amount of apoptotic cells determined. The mean and standard deviations of two independent experiments are shown. The numbers of apoptotic cells in the untreated controls were <10% and were subtracted from each experiment. In b, c, d and e, significant differences relative (rel.) to the untreated control cells (−) were determined using the two-tailed unpaired Student’s t-test (***P<0.001, **P<0.01).