Signaling Technology, Boston, MA, USA), or anti-b-actin (mouse monoclonal, cat. # A1978, Sigma-Aldrich, Saint Louis, MI, USA) antibodies, washed in TBS washing buffer and incubated with peroxidase-conjugated secondary antibodies. Enhanced chemilu- minescence reaction system (Roche) was used for visualization. MTT cell viability assay Cells were reseeded 24 hours after transfection into 3.9 cm2 dishes at 60,000 to 100,000 cells/well. After additional 24 h or 48 h of culture, medium was replaced by MTT-containing medium (thiazolyl blue, Carl Roth, Karlsruhe, Germany) and dishes were incubated for 2 h at 37uC. The MTT containing medium was replaced by solubilisation solution (10% Triton X- 100 (Carl Roth), 0.1 molar hydrochloric acid (Fisher Scientific, Schwerte, Germany) dissolved in isopropanol (Sigma-Aldrich)) and extinction measured at 570 nm. 48 h values were divided by 24 h values to correct for possible variations in numbers of seeded cells. BrdU cell proliferation assay DNA replication as a direct measure of mitotic activity was measured using the Cell Proliferation ELISA, BrdU chemilumi- nescence Kit (Sigma) according to the manufacturer’s protocol. Galectin-3 in Pancreatic Cancer Briefly, 5,000 to 10,000 cells were reseeded 24 hours after transfection into a 96 well mClear plates (Greiner Bio-One, Frickenhausen, Germany). BrdU containing medium was added for 4 or 6 h. After removal of the BrdU containing medium, cells were fixed for 1 h and subsequently incubated with the peroxidase-conjugated anti-BrdU antibody for 1.5 h. The chemi- luminescence reaction was measured in relative light units per second (rlu/s). Trail induced apoptosis In order to extrinsically induce apoptosis in PaTu 8988s cells, 300,000 cells were seeded in 9.6 cm2 culture dishes and treated with Trail protein (R&D Systems, Minneapolis, MN) at a dosage of 25 ng/ml for 24 h. Cell migration assay Modified Boyden chamber assays were performed by reseeding 20,000 to 40,000 cells resuspended in serum free media into transwell inserts with a pore size of 8 mm (BD Biosciences, Heidelberg, Germany). Inserts were suspended in 24well plates filled with medium supplemented with 1% FCS. Cells were allowed to migrate towards the lower face of the membrane for 2 Figure 3. No effect of Gal-3 knockdown or overexpression on pancreatic cancer cell viability in MTT assay. Transiently Gal-3 silenced cell lines (A), stably Gal-3 silenced S2-007 cell clones (B) or stably Gal-3 overexpressing PaTu 8988t cell clones (C) were cultured for 24 and 48 h followed by incubation with MTT reagent. 48 h values were divided by 24 h values (to correct for possible variations in numbers of seeded cells) and normalized to control siRNA transfected cells (NC). Values of stable Gal-3 knockdown cell clones (shGal-3 1, 2 and 3) and stable Gal-3 overexpressing cell clones (pGal-3 1, 2 and 3) were normalized to the mean of control vector transfected stable cell clones (shC 1, 2, 3 and pC 1, 2) respectively. Data includes a minimum of three independent experiments. doi:10.1371/journal.pone.0020859.g003 PLoS ONE | www.plosone.org 4 June 2011 | Volume 6 | Issue 6 | e20859