Fig. 6. Tcf4, ß-catenin and Smad1 positively regulate Myc. (A,B) Effect of RNAi-mediated decrease in Tcf4, ß-catenin or Smad1 on endogenous Myc mRNA and protein in mIMCD-3 cells. In control cells transfected with an empty plasmid, Bmp2 significantly increased levels of endogenous Myc mRNA and protein. (A,B) Knock-down of Tcf4 or ß-catenin decreased basal levels of Myc mRNA and protein, and totally blocked Bmp2-mediated increases in these species. Knock-down of Tcf4 exerted a larger inhibitory effect on basal levels. Knock-down of Smad1 did not exert a significant effect on basal mRNA and protein levels but did partially block the Bmp2 mediated increase observed in controls. (C) Effect of RNAi-mediated decrease in Tcf4, ß-catenin or Smad1 on luciferase activity downstream of the -1490 to -1 segment of the Myc promoter. In controls transfected with an empty RNAi-inducing plasmid, Bmp2 markedly induced luciferase activity. Knock-down of Tcf4 significantly decreased basal luciferase activity and totally abrogated the Bmp2-dependent increase in luciferase activity. Knock-down of either ß-catenin or Smad1 exerted no significant effect on luciferase activity under basal conditions. Knock-down of ß-catenin blocked the Bmp2-dependent increase. By contrast, the response to Bmp2 was limited to a partial but significant response in cells with a quantitatively similar reduction in Smad1 levels. (D) Role of TBE-A and SBE-A in Bmp2-dependent induction of Myc luciferase. mIMCD-3 cells were transfected with plasmid DNA encoding luciferase downstream of either wild-type or mutant forms of the Myc promoter (-1490 to -1). Under basal conditions (no Bmp2 treatment), luciferase activity was significantly decreased under the control of the mutant TBE-A and significantly increased downstream of the mutant SBE-A. Mutant TBE-A significantly decreased Bmp2-dependent induction of luciferase activity. Mutant SBE-A also decreased Bmp2-dependent induction of luciferase activity but not to the same extent as mutant TBE-A.