Figure 2.

DP-4 downregulates E2F site-dependent transcription. (a) U2OS cells were transfected with the indicated expression vectors (1 μg) as described and the effect on Apaf1-luciferase, Cdc6-luciferase, p73-luciferase and cyclinE-luciferase was measured. The luciferase activity is shown (relative to co-transfected pCMV-βgal expression), and lower panels show the level of ectopic proteins for each experiment. (b) U2OS cells were treated with non-targeting (NT) or DP-4 siRNA as described followed by immunoblotting; actin served as the loading control. (c) U2OS cells were transfected with DP-4 or NT control siRNA (25 nM) as described. After 72 h, cells were harvested, RNA extracted, reverse transcribed and the level of Cdc2, Apaf1, p73 and GAPDH RNA was assessed. (d) U2OS cells were transfected with DP-4 or NT control siRNA (25 nM) as described. After 72 h, cells were harvested and extracts immunoblotted with antibodies against Cdc2, Apaf1, DHFR, E2F-1 and actin