Api5 depletion induces a decrease in E2F1-target gene mRNA levels without affecting E2F1 expression.

<p><b>A.</b> Api5 and E2F1 reciprocal regulation. After depletion of Api5 and E2F1 with specific siRNAs, H1299 cells were made quiescent by serum starvation for 48 h (0% FBS) and then allowed to progress through the cell cycle over 10 hours by FBS addition (10 h refeeding). Control cells were grown in 10% fetal bovine serum media (10% FBS). Api5, E2F1 and β-actin expression was analyzed by Western blot (WB). <b>B.</b> E2F1 and β-actin expression was analyzed by Western blot (WB) after Api5 overexpression. HeLa cells were transfected with different amounts of a plasmid encoding Api5. <b>C.</b> Api5 and E2F1 immunocytochemistry. HeLa cells were transfected with the indicated siRNAs or treated with etoposide for 16 hours. Endogenous immunodetection of Api5 (green) and E2F1 (red). Nuclei were stained with PI (blue). [Scale bar 20 µm]. <b>D.</b> Retinoblastoma expression under Api5 overexpression or depletion. Hela cells were transfected or not with a plasmid encoding Api5 or an siRNA directed against Api5. Western blot analysis was performed with an anti-retinoblastoma antibody. <b>E.</b> siRNA efficiency of quantitative PCR samples. HeLa cells were treated with Api5 or E2F1 siRNAs. Api5 and E2F1 protein content was assessed by Western blot (left panel) to evaluate siRNA efficiencies by densitometric analysis (relative to scrambled treated cells) (right panel). <b>F.</b> E2F1 target gene expression. HeLa cells were treated with Api5 or E2F1 siRNA, or with both siRNAs. Api5, E2F1, cyclin E, cyclin A, cyclin D1, p16 INK4a, Cdk2 and Cdc25A relative mRNA levels were measured by quantitative PCR, using HPRT1 and GUSB mRNA levels as references. Asterisks indicate a significant decrease compared to control conditions (scrambled siRNA treated cells). Statistical significance was determined by two-tailed Student’s <i>t</i> test (*, p<0.05). <b>G.</b> Cyclin A expression. Western blot analysis of cellular levels of cyclin A protein after transfection of H1299 cells with Api5 or E2F1 siRNAs.