FIG. 8.

Induction of endogenous ZTA and EAD protein expression by C/EBPα exogenously introduced into a B-lymphoblast cell line latently infected with EBV. (A) Introduction of Flag-C/EBPα encoded by pSEW-C02 into electroporated EBV-infected Akata cells. (Upper panel) Double-label IFA showing the lack of Flag-protein expression from empty vector control DNA, detected with an anti-Flag MAb (FITC, green) (left); the absence of enhanced ZTA protein expression in the same cell population, detected with an anti-ZTA PAb (rhodamine, red) (middle); and stained nuclear DNA in the whole-cell population (DAPI, blue) (right). (Lower panel) Double-label IFA showing the expression of Flag-C/EBPα from plasmid pSEW-C02, detected with an anti-Flag MAb (FITC, green) (left); the activation of ZTA protein expression in two out of four visible C/EBPα-positive cells, detected with an anti-ZTA PAb (rhodamine, red) (middle); and a merge of the two frames (yellow) (right). Induced ZTA expression was found to occur in 28% of Flag-C/EBPα-positive cells. (B) Activation of EBV EAD (PPF or BMLF1) protein expression by the introduction of exogenous Flag-C/EBPα plasmid DNA (pSEW-C02) into EBV-infected Akata cells by electroporation. All three sets of panels show examples of induced EAD expression detected by double-label IFA: EAD-expressing cells, detected with a mouse anti-EAD MAb (FITC, green) (first set of panels); C/EBPα-expressing cells, detected with a rabbit anti-Flag PAb (rhodamine, red) (second set of panels); a merge of those two sets of panels (yellow) (third set of panels); and staining of all nuclei in the same fields (DAPI, blue) (fourth set of panels). Induced EAD expression was found to occur in 85% of Flag-C/EBPα-positive cells.