Galectin-3 in Pancreatic Cancer time PCR (qRT-PCR) was performed using SYBR Green MasterMix (Applied Biosystems, Wellesley, MA, USA) and specific primer pairs designed with the PrimerExpresss program (Applied Biosystems). The following primer pairs were used for qRT–PCR: ribosomal protein, large, P0 (RPLP0) fwd: 59-TGGGCAAGAA- CACCATGATG-39; rev. 59-AGTTTCTCCAGAGCTGGGT- TGT-39; Galectin-3 fwd: 59-AGAGGGAATGATGTTGCC- TTCC-39; rev: ACAATGACTCTCCTGTTGTTCTCATT-39. Subcellular fractionation and immunoblotting Subcellular fractionation was performed as described previously [33]. Briefly, cells were washed twice with icecold PBS and collected by centrifugation at 1.600 r.p.m. at 4uC. Lysates were then resuspended in buffer A (10 mM Hepes pH 7.9; 10 mM KCl; 0,1 mM EDTA; 0.1 mM EGTA; 1 mM DTT; proteinase inhibitors) for 15 min and subsequently centrifuged for 20 min at 3.600 r.p.m. Supernatants were transferred to new cups and centrifuged at 14,000 r.p.m. for additional 4 min. Pellets were resuspended in 30–100 ml buffer C (20 mM Hepes pH 7.9; 0.4 M NaCl; 1 mM EDTA; 1 mM EGTA; 1 mM DTT; proteinase inhibitors) and incubated on ice for 30 min. A final centrifugation step at 14,000 r.p.m. for 10 min was performed to separate nuclear proteins from cellular debris. For Western blotting, the resulting nuclear protein extracts were electrophoresed through a 7.5% SDS–polyacrylamide gel and transferred onto PVDF Immobilon-P membranes (Millipore, Billerica, MA, USA) as described previously [34]. Membranes were probed with either anti-Gal-3 (mouse monoclonal, cat. # A3A12, abcam, Cam- bridge, UK), anti-ORC2 (polyclonal rabbit, cat. # 559266, BD Biosciences), anti-PARP (rabbit polyclonal, cat. # 9542, Cell Figure 2. Silencing and overexpression of Gal-3 in pancreatic cancer cell lines. (A) Transient knockdown of Gal-3 was performed by transient transfection of multiple cell lines using two different Gal-3 specific siRNA sequences (siGal-3 1 and 2) or a non-silencing control siRNA (NC). (B) Stable knockdown in S2-007 was performed using shRNA expression constructs. Three Gal-3 specific shRNA transfected clones (shGal-3 1, 2 and 3) and three nonsilencing control shRNA transfected clones (shC 1, 2 and 3) were chosen for further analysis. (C) Stable overexpression of Gal-3 was achieved by transfection of PaTu 8988t cells with a Gal-3 expression vector (pGal-3 1, 2 and 3) or a control vector (pC 1 and 2). U denotes untransfected cells. Total cell lysates were analyzed by qRT-PCR (upper panels) and Western blot (lower panels) for Gal-3 expression levels. Gal-3 mRNA levels were determined relative to RPLP0. siGal-3 1 and 2 were normalized to NC. b-actin was used as loading control for the Western blots. doi:10.1371/journal.pone.0020859.g002 PLoS ONE | www.plosone.org 3 June 2011 | Volume 6 | Issue 6 | e20859