While Smad2 phosphorylation duration correlates to TGF-β and Smad7 expression, cytostatic responsiveness relates to CAGA-reporter-activation.

<p>HCC cell lines were treated with 5 ng/ml TGF-β for 0, 1, (3), 7, 24 and 48 h. (A) Left side: Western blot analysis of Smad2 phosphorylation and GAPDH expression. Right side: To evaluate TGF-β dependent transcriptional activity of Smad2, cells were transfected with plasmids carrying a luciferase gene under control of the activin response element (ARE) and additionally with a FAST-1 expression construct. Luciferase activity of cells treated with 5 ng/ml TGF-β for 9 h was correlated to untreated control samples. (B) Left side: Western blot analysis of Smad1/3 phosphorylation (1: upper/3: lower band); loading control GAPDH. Right side: TGF-β dependent transcriptional activity of Smad3 was evaluated 9 h after TGF-β treatment in cells infected with an adenovirus carrying a luciferase gene controlled by a CAGA response element. Treated samples were correlated to the corresponding untreated control. In the table, cell lines marked as black, show TGF-β induced cell death and growth inhibition whereas cells marked as grey mainly react with the latter one. The gradient from white to black displays increasing basal Smad7 expression levels. (C) Immunofluorescent detection of linker phosphorylated Smad3 in uninduced HCC-T cells. Red fluorescence indicates nuclear localization of pSmad3L. Blue staining indicates nuclei stained with DRAQ5. Significant differences are indicated as * p < 0.05, ** p < 0.01 and *** p < 0.001 (Student’s t test).