Figure 3

Cluster lysine 161 and 164 are important for E2F1 activity. (a and b) Gene luciferase experiments performed in Hela cells transfected with CCNE promoter-Firefly luciferase reporter plasmid and pCMV-3HA-E2F1 wt or E2F1 mutants constructs in which indicated K have been mutated into R (a) or (b) K0 (=mutation of every K into R), K89 only (=mutation of every K into R except K89), K89, 137 (=mutation of every K into R except K89 and K137) or K89, 137, 161, 164 (=mutation of every K into R except K89, K137, K161, K164). Luciferase activity was normalized to β-galactosidase activity. Results are expressed as % of activity measured with E2F1 wt. Mean±S.D. of at least three independent experiments. Statistical analysis performed using Student’s t-test. ***P<0.001, **0.001<P<0.01, *0.01<P<0.1, NS P>0.1. The expression of the constructs was checked by a western blot analysis (upper panel). β-Actin was used as loading control. (c-f) U2OS cells were transfected for 48 h with empty pCMV-3HA vector (V), pCMV-HA-E2F1 or pCM-3HA-E2F1 K161/164R mutant. (c) Western blot analysis of E2F1, 48 h after transfection. β-Actin was used as loading control. (d) Relative expression of ccne, tp73 and apaf1 mRNA as measured by RT-qPCR 48 h after transfection. Results were normalized to hprt mRNA and expressed relative to cells transfected with empty vector. Mean±S.D. of three independent experiments. (e) Chromatin IP experiments were performed using an anti-E2F1 or an irrelevant antibody (IgG). The levels of E2F1-associated genomic DNA region encompassing E2F1 binding site of Cyclin E (CCNE), TP73 or APAF1 promoter were quantified by qPCR. Results were normalized to input and the recruitment with irrelevant antibody, and expressed as relative recruitment compared with cells transfected with empty vector. (f) Proliferation of U2OS cells transfected for 48H with empty vector, E2F1 or E2F1 K161/164R mutant, as analyzed by crystal violet staining. Mean±S.D. of three independent experiments. Statistical analysis performed using Student’s t-test. ***P<0.001, **0.001<P<0.01, *0.01<P<0.1, NS P>0.1