Figure 1.

Effects of dn-E2F on cell growth. (A) The E2F1 and DP1 expression vectors (0.6 μg) were cotransfected into 60-mm plates of SVts8 cells along with a reporter plasmid and lacZ plasmid. Where indicated, cells were also cotransfected with an increasing amount of expression plasmid encoding dn-E2F (lane 2: 0 μg; lane 3: 0.1 μg; lane 4: 0.3 μg; lane 5; 0.6 μg). Error bars indicate SD. (B) Early passage (45PDLs) TIG-3 cells infected with retrovirus encoding dn-E2F or empty vector. Cells were analyzed for expression of E2F1 and dn-E2F protein by Western blot after selection with hygromycin. The antibody against β-actin was used as a loading control. (C) EMSAs were performed using a radiolabeled probe containing the E2F consensus sequence of the adenovirus E2 gene promoter and extracts from cells infected with retrovirus encoding empty vector (lanes 1–5) or dn-E2F (lanes 6–10) in the absence or presence of antibodies as indicated. The DNA-binding activity, which is not shifted by any available E2F antibodies, was marked by an asterisk. (D and E) Cell extracts were prepared from cells at 60PDLs and subjected to RT-PCR (D) or Western blotting (E) with primers or antibodies shown at left. E2F target genes reported previously were highlighted with an asterisk. CDK4 was used here as a loading control. (F) Early passage (45PDLs) TIG-3 cells infected with retrovirus encoding dn-E2F or empty vector were selected for expression of the hygromycin selectable marker for 5 d and used in proliferation curves performed in triplicate. Error bars indicate SD.