Figure 8.

Repressor function of EBNA5 does not correlate with the induction of cell death. DG75 cells were co-transfected with the CD2 expression vector pE300CY6, the pSVECAT reporter plasmid and increasing amounts of the EBNA5 expression vectors pCI-EBNA5 (A) or pcDNA6-EBNA5 (B and C). The transfected cells were selected using anti-CD2 antibody-coated magnetic beads, plated and assayed for EBNA5 expression, CAT reporter gene expression, luciferase reporter gene expression, Trypan Blue exclusion (cell viability), [³H]thymidine incorporation (DNA synthesis rate) and staining with Annexin V–FITC (apoptosis) and propidium iodide (necrosis), respectively, as described in Materials and Methods. The results of three independent experiments are presented as the means ± SEM. (A) Trypan Blue exclusion, [³H]thymidine incorporation and CAT reporter gene expression at different EBNA5 levels. The observed values of each parameter are expressed as a percentage of the value obtained in the absence of EBNA5 expression. Thus, the 100% value corresponds to the [³H]thymidine incorporation rate, the fraction of cells excluding Trypan Blue or the CAT activity in cells transfected with 540 fmol empty pCI plasmid. The means ± SEM of three independent experiments are presented. EBNA5 expression in the transfected cells was determined by dot immunoblot analysis using the anti-EBNA5 antibody JF186. Cell extract corresponding to 90 000 transfected cells was applied in each dot. The amount of pCI-EBNA5 DNA per 5 × 10⁶ cells used in the transfections is indicated in the figure. (B) Frequency of apoptosis and necrosis of EBNA5-expressing cells. Aliquots of transfected and selected cells were incubated with Annexin V–FITC, with propidium iodide and with a mixture of Annexin V–FITC and propidium iodide, respectively. The fractions of Annexin V–FITC (early apoptosis) and propidium iodide (necrosis and late apoptosis) stained cells were determined by cell sorting of 5000 cells with a FACScan apparatus. The means ± SEM of three independent experiments are presented. (C) The activity of the pCMVLuc reporter plasmid in the cells in (B). The means ± SEM of three independent experiments are presented.