FIGURE 2.

HMGA2 and Smad3/Smad4 cooperate to activate the Snail1 promoter. A, luciferase reporter assays of the Snail1 promoter construct in HepG2 cells transiently transfected (+) or not (-) with an HA-HMGA2 expression construct and treated (+) or not (-) with 1 ng/ml TGF-β1 for 24 h. B, luciferase reporter assays of the Snail1 promoter construct in HepG2 cells transiently transfected with FLAG-Smad3, FLAG-Smad4, and HA-HMGA2 expression constructs as indicated. Levels of expression of the transfected proteins were assessed by immunoblot using anti-FLAG and anti-HA antibodies. Stars indicate nonspecific bands. C, immunoblot analysis of endogenous SNAIL1 in HepG2 cells transiently transfected with FLAG-Smad3, FLAG-Smad4, and HA-HMGA2 expression constructs as indicated. α-Tubulin served as a loading control. D, luciferase reporter assays of the indicated deletion constructs of the Snail1 promoter in HepG2 cells transiently transfected with FLAG-Smad3, FLAG-Smad4, and HA-HMGA2 expression constructs as indicated. A schematic representation of potential binding elements for different transcription factors present in the Snail1 promoter and the breakpoints of the deletion constructs used are shown. In all panels normalized luciferase data are plotted in bar graphs as averages with S.D. derived from triplicate determinations.