SMAD4 reduces E-cadherin, VEGF, EGFR and CD133 expression, but increases TGFβ1/Nestin and CD44 protein levels in PDAC. (A) SMAD4 modulates mRNA levels of EMT and CSC markers in PDAC cells. RT qPCR analyses were performed in AsPC-1 and PANC-1 SMAD4 deficient or proficient cells. Compared to the control, restoration of SMAD4 reduces mRNA level of EGFR, VEGF, CD133 and E-cadherin, but increased Vimentin, SMA, Nestin and CD44 mRNA expression in PDAC. Data were means ± SD of triplicates. *P < 0.05 (B) Western blot analysis of three PDAC cell lines, which had overexpression or knockdown for SMAD4 revealed that the expression levels of CD133, Nestin, EGFR, VEGF and EMT markers in indicated cell lines. β-actin was included as a loading control. (C) Luiferase activity assays for the analysis of CD133 and Nestin transcriptional activities in PDAC cells. Reproter assays were performed using CD133-luc (a) and Nestin-luc(b) reporter constructs in AsPC-1, CFPAC-1 and PANC-1 Smad4 proficient and deficient cells. Bars represent means derived from at least 3 independent experiments. *P < 0.01.