Figure 2.
Activation of Siah1 promoter by E2F1. (A) Determination of minimal promoter region of Siah1 that can be activated by E2F1. H1299 cells were individually cotransfected with equal amount (100 ng) of eight indicated Siah1 promoter deletion constructs of Luc‐reporter, together with either pcDNA3 empty vector (200 ng) or E2F1 expressing vector pcDNA3‐E2F1 (200 ng). Renilla luciferase plasmid pRL‐CMV was also introduced into each transfected cell samples as internal control. Cells were harvested 24 hrs after transfection and luciferase activities were assayed using the dual luciferase assay system. Fold activation values were measured relative to the levels of Renilla luciferase activity and were listed under their respective bars. (B) pSiah‐BglII is sufficient to activate Siah1 promoter activity in response to E2F1. H1299 cells transfected with either reporter plasmids pSiah‐BglII (100 ng) or pApaf‐Luc (pGL3‐Apaf‐1[–396/+208])(100 ng) were further cotransfected in combination with pcDNA3 expression vectors (200 ng) or pcDNA3‐E2F1 (200 ng). Renilla luciferase plasmid pRL‐CMV was also introduced into each transfected cells as internal control. Fold activation values were measured relative to the levels of Renilla activity. (C) Schematic diagrams of E2F deletion mutants of Siah1 promoter. (D) H1299 cells were transfected with either reporter plasmids pSiah‐BglII (100ng) or one of E2F1 deletion mutants (pSiah‐BglII‐M1, pSiah‐BglII‐M2 and pSiah‐BglII‐MM, 100 ng) along with pcDNA3 expression vectors (200 ng) or pcDNA3‐E2F1 (200 ng). Renilla luciferase plasmid pRL‐CMV was also introduced into each transfected cells as internal control. Fold activation values were measured relative to the levels of Renilla activity. (E) Activation of Siah1 promoter by E2F1 is dose‐dependent. H1299 cells were cotransfected with pSiah‐BglII (200 ng), together with either pcDNA3 vector (200 ng), pcDNA3‐E2F1(1–363) (200 ng, an E2F1 deletion mutant) or increasing concentrations of pcDNA3‐E2F1 (from 50 ng, 100 ng to 200 ng) separately. Renilla luciferase plasmid pRL‐CMV was introduced into each cotransfection cell sample as internal control. Luciferase activity was measured and plotted. (F) Siah1 promoter specifically responds to E2F1. H1299 cells were cotransfected with reporter plasmid pSiah‐BglII (200 ng), together with one of the indicated activator plasmids: pcDNA3, pcDNA3‐E2F1, pcDNA3‐E2F2 and pcDNA3‐E2F3 at equal amount. Renilla luciferase plasmid pRL‐CMV was introduced into each cotransfected cell samples as internal control. Luciferase activity was measured and plotted after normalizing with respect to Renilla luciferase activity. In (A)–(F), all cells were harvested 24 hrs after transfection and Luciferase and Renilla activity assayed using the Dual‐Luciferase Reporter Assay System (Promega Corporation). Luciferase values were corrected for transfection efficiency with Renilla activity. All experiments were independently performed in triplicate. Vertical error bars are the standard deviations from the mean of the values within three standard deviations.