Figure 5

Effect of DFMO on E2F transcriptional activity. A: E2F electoromobility shift assays using 10 μg of nuclear extracts from DFMO treated Y79 cells and E2F binding oligonucleotides. Lanes 1–4 : Nuclear extracts from Y79 cells treated with 5 mM DFMO for 0, 24, 48 h or 5 mM DFMO and 20 μM putrescine for 48 h were incubated as described under Experimental. Lanes 5–7 : Nuclear extracts from Y79 cells treated with 5 mM DFMO for 24 h were preincubated with control IgG, E2F-4 antibody, and p107 antibody, respectively. Lanes 8, 9 : Nuclear extracts from Y79 cells treated with 5 mM DFMO for 24 h were incubated with 50 ng of unlabeled competitor (WT and MT, respectively). B: One μg of pE2WTx4-Luc and 10 ng of pRL-CMV were co-transfected into Y79 cells (5 × 10⁵) using FuGene™ Transfection Reagent. After 24 h culture media were changed to RPMI 1640 media with or without 5 mM DFMO or 5 mM DFMO and 20 μM putrescine. Cell lysates were prepared at 24 h, 48 h and 72 h after the addition of DFMO, and examined for luciferase activity to monitor the promoter activity. Significantly different compared to control cells: *p < 0.02