Figure 4 RGC-32 mediates TGF-β-induced EMT in BxPC-3 cells. BxPC-3 cells were transfected with RGC-32 siRNA (siRGC-32) or negative control siRNA (siCtrl). 6 h later, cells were starved in serum-free RPMI-1640 for additional 6 h, followed by treatment with or without 10 ng/ml TGF-β1

for 72 h. The mRNA expression and protein expression of RGC-32, E-cadherin and vimentin were examined by qRT-PCR (A) and western blot (B and C) respectively. β-actin was used as an internal control. RGC-32 silencing significantly blocked TGF-β-induced EMT in BxPC-3 cells. *P < 0.05. RGC-32 mediates TGF-β-induced migration of BxPC-3 cells We used transwell cell migration assay to examine the role of RGC-32 in cell migration of BxPC-3 cells. As shown in Figure 5, TGF-β treatment promoted the migration of BxPC-3 cells while RGC-32 RNA silencing P005091 remarkably blocked this effect, implicating that RGC-32 mediated TGF-β-induced migration of BxPC-3 cells. Figure 5 RGC-32 mediates TGF-β-induced migration of BxPC-3 cells. BxPC-3 cells were transfected with siRGC-32 or siCtrl and treated with 10 ng/ml TGF-β1 or not as described before. 24 h later, 2 × 105 CAL-101 chemical structure cells were loaded into the top chamber of 24-well transwell plates and incubated for

another 24 h. The migrated cells were stained with 0.1% crystal violet and the average number per field was quantified under high power (original magnification × 200) of the phase contrast microscope. *P < 0.05. Discussion Recent studies have implicated EMT in cancer progression by showing that epithelial-like tumor cells could switch to a mesenchymal-like phenotype that facilitates motility and invasion [21]. EMT-related L-NAME HCl molecular pathways have been extensively investigated, and various genes and molecules have been identified as important factors in EMT, of which TGF-β has been most studied and believed to be the major inducer in pancreatic cancer [22]. It has

been demonstrated that when TGF-β binds to the TGFβRII, TGFβRI becomes phosphorylated and propagates the signal downstream through phosphorylation and thereby activation of the Smad2 and Smad3 proteins (receptor Smads). The activated receptor Smads form a complex with Smad4 and translocate into the nucleus to regulate the expression of genes involved in EMT [23, 24]. Beside Smad-mediated transduction, TGF-β also induces EMT via Smad-independent signaling cascades Selleck AMN-107 including PI3K, MAPK, Rho kinase pathways and so on [25]. Our research demonstrated that constant stimuli by TGF-β induced EMT in BxPC-3 cells and the observed changes were proposed to be independent of Smad pathway because Smad4 is homozygous deleted in BxPC-3 cells [26]. The result was consistent with that in Vogelmann R et al’s research [9]. However, the downstream effectors of Smad-independent pathways mediating TGF-β-induced EMT remain largely unknown.