pancreatic cancer cells have not been reported. The purpose of this study was thus to experimentally evaluate the effects of overexpression or knockdown of Gal-3 in a comprehensive set of pancreatic cancer cell lines (PaTu 8988s, PaTu 8988t, S2-007, S2- 028, IMIM-PC-1 and MIA PaCa-2). Functional analyses included assays for cell viability, apoptosis, proliferation, migration and anchorage independent growth as well as tumor growth in a xenograft mouse model. Apart from isolated effects in single cell lines, modulation of Gal-3 expression had no consistent effect on tumor-relevant characteristics of pancreatic cancer cells. Materials and Methods Human tissues and cell lines The human pancreatic adenocarcinoma cell line IMIM-PC-1 [31] was kindly provided by F.X. Real (Insitute Municipale de Investigacion Medica, Barcelona, Spain). S2-028 and S2-007 [32] were from T. Iwamura (Miyazaki Medical College, Miyazaki, Japan). MIA PaCa-2 was obtained from the American Type Culture Collection (ATCC, RMD, USA). PaTu 8988t and PaTu 8988s were kindly provided by H.P. Elsa¨sser (Institut fu¨r Klinische Zytobiologie und Zytopathologie, Philipps Universita¨t, Marburg, Germany). All cell lines were maintained in Dulbecco’s modified minimal essential medium (GIBCO, Invitrogen Corp., NY, USA) supplemented with 10% FCS (GIBCO, Invitrogen Corp., NY, USA) and Gentamicin 0.045 mg/ml (GIBCO, Invitrogen Corp., NY, USA). Ethics Statement Surgically resected pancreatic adenocarcinoma and chronic pancreatitis tissues were provided by the surgery departments at the Universities of Ulm and Homburg/Saar. Normal pancreas samples were obtained from healthy areas at the borders of chronic pancreatitis resectates. Written informed consent was obtained from all patients prior to using tissue samples. The study was approved by the ethics committee at the University of Ulm, Germany (Ethikkommission der Universitaet Ulm) as well as the ethics committee at the University of Homburg/Saar, Germany (Ethikkommission der Universitaet Homburg). Galectin-3 in Pancreatic Cancer Transfection of cell lines Small interfering RNA (siRNA) was transfected into PaTu 8988s, S2-007 and S2-028 cells using siLentFect Lipid Reagent (Bio-Rad, Munich, Germany) according to the manufacturer’s protocol. SiRNA transfection into MIA PaCa-2 cells was performed using Transmessenger reagent (Qiagen, Hilden, Germany) and IMIM-PC-1 cells were transfected with X- tremeGENE siRNA Transfection Reagent (Roche, Mannheim, Germany) according to the manufacturers’ protocols, respectively. The Gal-3-specific siRNAs were: siGal-3-1, Hs_LGALS3_1 FlexiTube siRNA SI00470036 and siGal-3-2, Hs_LGALS3_2 FlexiTube siRNA SI00470043 (Qiagen). Silencer Negative Control from Ambion was used as non-silencing control. The Gal-3 expression vector was constructed by cloning the PCR-amplified Gal-3 open reading frame into the pcDNA V3.2/ V5 dest vector using the Gateway recombination cloning technology (Invitrogen Life Technologies, Karlsruhe, Germany). Following transfection of PaTu 8988t cells using Lipofectamine 2000 Transfection Reagent (Invitrogen), selection of stably transfected cell clones was performed by adding 800 mg/ml G418 to the culture medium. A Gal-3-specific shRNA expression construct in the pGIPZ vector was purchased from Open Biosystems, Huntsville, AL, USA (cat. # RHS4430-99137619). Non-silencing shRNAmir (cat. # RHS4348, Open Biosystems) was used as the negative control. Stable transfection of the S2-007 cells was performed using Lipofectamine 2000 (Invitrogen). Stably transfected clones were selected by adding hygromycin (400 mg/ml) to the culture medium. RNA Extraction and qRT-PCR RNA from cell lines was extracted using peqGold Total RNA Kit (PEQLAB Biotechnologie GmbH, Erlangen, Germany) according the manufacturer’s protocol. Bulk tumor tissue samples were homogenized on dry ice/liquid nitrogen using a mortar and pestle. RNA was extracted using the RNeasy Mini Kit (Qiagen) according the manufacturer’s protocol. First-strand cDNA was synthesized from 1 mg total RNA using the Omniscript RT Kit (Qiagen) according to manufacturer’s protocol. Quantitative real Figure 1. Overexpression of Gal-3 in pancreatic cancer primary tissues and cell lines. Gal-3 mRNA levels were determined by quantitative real-time PCR (qRT-PCR) (A, B). Ribosomal protein, large, P0 (RPLP0) was used as the reference gene. PC: pancreatic cancer ; CP: chronic pancreatitis ; NP: normal pancreas. Protein expression in different pancreatic cancer cell lines was determined by Western blot (C) analyses. b-actin was used as loading control. doi:10.1371/journal.pone.0020859.g001 PLoS ONE | www.plosone.org 2 June 2011 | Volume 6 | Issue 6 | e20859