Incorporation of the TSTA system into a single plasmid. Incorporation of the TSTA system into a single plasmid. A, a representation of the two different single TSTA plasmids assayed for activity. Left, representative of the forward variant; right, representative of the reverse variant. B, the forward and reverse variants shown in A were tested for their luciferase activity following titration into 293T cells. As a control, CMV-driven VP16-E2 (pCG-VP16-E2) activating transcription from p6xE2-3bp-Luc was included. C, a comparison of luciferase activity generated with the forward plasmid shown with that generated by the TERT promoter directly driving expression of luciferase. One microgram of each plasmid was used in 293T cells. D, a comparison of luciferase activity generated by E2 versus Gal4 in identical one-plasmid systems. All transcription results are presented as fold activation over the activity generated by the luciferase reporter by itself and are normalized to protein concentration. All experiments were carried out at least three times in duplicate. Bars, SE. The P values shown represent the result of t test analysis on at least three independent experiments. Maja L. Arendt et al. Mol Cancer Ther 2009;8:3244-3254 ©2009 by American Association for Cancer Research