FIG. 4.

Proliferative activities of mutant EBNA2 alleles. Specific mutants of EBNA2 were placed into the pAG138 vector (11) by removal of the appropriate sequences from the various pSG5-EBNA2 alleles. This was done by partial digestion of the pSG5 mutant construct with PstI, blunt-end formation, and subsequent complete digestion with BstEII. Fragments of appropriate size were then ligated into pAG138 previously digested with SmaI and BstEII. The alteration was verified by sequence analysis. Transfection of plasmids and transduction of EREB2.5 cells were performed as previously described (11). The transduced cells were cultured in the presence of 1 μM estradiol for 5 days, after which they were transferred to estrogen-free medium for 4 weeks. Proliferation was determined by reduction of Alamar blue according to the manufacturer's instructions (BioSource). For each transduced culture, a proliferative index was established by determining the least-squares fit of the time course of the measured percentage of Alamar blue reduction for a fixed number of cells (5 × 10⁴ cells/well) in a 96-well plate over a 48-h period. The ordinate indicates the relative proliferation compared with pAG138-transduced cells in this assay. All assays were carried out in triplicate. The complete assay was performed on three separate occasions, using separate plasmid preparations for all of the components of the assay. Each error bar indicates the standard deviation for the three different determinations.