Figure 3. TRADD was involved in LMP1-induced cell death.

(A) MDCK cells were transfected with an empty vector or a vector expressing LMP1. Cells were then incubated with propidium iodide (PI) before analysis of PI labelling using flow cytometry. (B) MDCK cells were co-transfected with pRLnull and LMP1 plasmids. Cell viability was determined by a luciferase activity assay. Luciferase activity of cells transfected with empty vectors was indexed to 100%. Values represent the means of triplicate assays ± SD from a representative experiment. Experiments were performed three times and similar results were obtained. (C) MDCK cells were co-transfected with the pRL null plasmid, the LMP1 expressing plasmid (1 µg DNA) and increasing doses (0, 0.5, 1 µg DNA) of plasmids expressing dominant negative versions of TRADD protein (TRADD mut) or TRAF2 (TRAF2 mut). Cell viability was determined by assessing the activity of Renilla luciferase. The luciferase activity of control cells transfected with empty vectors was indexed to 100%. Values represent the means of triplicate assays ± SD of a representative experiment. Experiments were performed three times and similar results were obtained. Whole cell extracts were resolved by 10% SDS-PAGE and analyzed by Western blot to assess gene expression, using antibodies directed against LMP1, TRADD or TRAF2. Actin was used as a loading control. (D) Cells were co-transfected with a vector in which five elements responding to NFκB control the expression of the firefly luciferase gene, a LMP1 expressing vector and vectors expressing dominant negative versions of TRADD protein (TRADD mut) or TRAF2 (TRAF2 mut). The luciferase activity of control cells transfected with empty vectors was indexed to 1.