Generated stable SMAD4 rerexpressing or knockdown of SMAD4 in human PDAC cells. (A) Western blot analysis indicated that SMAD4 was successfully restored or knocked down in human PDAC cells as compared to control cells. β-actin was used as an internal control. (B) AsPC-1, CFPAC-1 and PANC-1 SMAD4 proficient and deficient cells were transiently transfected with SBE4 luciferase reporter and Renilla luciferase constructs. Cells were treated under the indicated conditions for 24 hours. Luciferase reporter assays were conducted using a dual luciferase assay, and Renilla luciferase activity was used as an internal control. Mean + SE (n = 3) *P <0.01. (C) Western blot detection of total and phosphorylated SMAD2 in SMAD4 proficient or deficient AsPC-1 and PANC-1 cells with or without TGF-β (10 ng/μl). Result confirmed TGF-β increased phosphorylation of SMAD2 in the SMAD4 restoration cell lines. β-actin was used as an internal control. (D) Immuno- fluorescence analysis confirmed that SMAD4 expression in AsPC-1 SMAD4 cells and mock control cells, and the nuclear localization of SMAD4 was observed in response to TGFβ1 treatment (magnification ×100).