Fig. 6. Increased E2F activity is associated with elevated levels of E2F-mediated transcription. (A) The CAT expression plasmids E2F1CAT and 4XE2FCAT under E2F-dependent promoters as well as ß-galactosidase control constructs were co-transfected into HuH-7 cells as described in Materials and Methods. 24 hours post-transfection, the cells were cultured in the presence (+) or absence (-) of 1 nM TGF-ß1 for an additional 36 hours before harvesting the cells for determination of CAT and ß-galactosidase activity. (B) The E2F-controlled CAT constructs were co-transfected into the cells with (+) or without (-) pCMV.RB expression plasmids. After 24 hours, TGF-ß1 was added to the incubation media and the cells harvested for CAT analysis as indicated above. The results, normalized to ß-galactosidase expression, are the mean determinations of at least three different experiments, each done in duplicate.