Figure 5.

E2F1 DNA-binding, transactivation, and pRb-interaction are required for TGFβ-mediated apoptosis. HuH7 cells transiently transfected with empty vector or mutant E2F1 expression constructs as indicated were untreated or treated with 100 pM TGFβ for 24 h. (a) Cell viability was assessed by calcein-AM assay, with bars representing means±S.D. (b) Caspase 7 and Smac/DIABLO mRNA levels were measured by real-time qPCR analysis. Results are normalized to GAPDH and show the mean±S.D., expressed as relative to levels observed in untreated cells (set to 1). (c) HuH7 cells untreated or treated with TGFβ (100 pM) were subjected to immunoprecipitation (IP) with the specified antibodies followed by western blotting (WB) to assess levels of associated E2F1 and pRb