Figure 8.

Mel-18 and c-Myc regulate Bmi-1 promoter activity. (A) Overexpression of Mel-18 down-regulates only the wild-type Bmi-1 promoter. pGL3-Bmi-1 PrWT, pGL3-Bmi-1PrMut, and pGL3- Bmi-1PrΔMyc plasmids were transiently transfected into 293T cells together with an increasing amount of Mel-18–overexpressing plasmid (pLPC-Mel-18) and a plasmid expressing renilla luciferase. Forty-eight hours after transfection luciferase activity was determined as described in Materials and Methods. (B) Transient overexpression of c-Myc up-regulates wild-type Bmi-1 promoter activity through the c-Myc binding site. Different promoter-reporter constructs (as indicated) were transiently transfected into 293T cells with an increasing amount of pCMV-Myc expression plasmid together with a plasmid expressing renilla luciferase, and luciferase activity was determined as described in Materials and Methods. (C) c-Myc knockdown using transient transfection of a plasmid containing c-Myc shRNA down-regulates activity of the Bmi-1 promoter, which contains an intact c-Myc binding site. The promoter activity of various promoter-reporter constructs with the increasing amount of a plasmid expressing c-Myc shRNA was analyzed in 293T cells as described in Materials and Methods.