Galectin-3 in Pancreatic Cancer Figure 4. Inconsistent effect of Gal-3 on cell proliferation as measured by BrdU uptake. 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 with BrdU agent for 2 or 4 h. BrdU incorporation by proliferating cells was measured by ELISA. Values of cells transiently transfected with different Gal-3 specific siRNAs (siGal-3 1 and 2) were 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. * Indicates p,0.05 as compared with control siRNA transfected cells (double-sided unpaired t-test). doi:10.1371/journal.pone.0020859.g004 or 4 h at 37uC. Non-migrated cells were removed from the inside of the inserts using cotton swabs. Migrated cells were stained by submerging the membranes in 0.2% crystal violet dissolved in 20% methanol for 10 min. Migrated cells were counted using light microscopy at a magnification of 106. Soft agar assays In order to assess the potential for anchorage-independent growth, soft agar assays were performed as described previously [35]. In brief, 16104 cells per 9.6 cm2 cell culture dish were seeded in DMEM/0.33% bacto-agar onto a bottom layer of DMEM/0.5% bacto-agar. Anchorage-independent growth was measured after 7 days of incubation by counting the number of viable colonies. Nude mouse xenografts NMRI-nu/nu mice were propagated and maintained in a pathogen-free environment. Female 6–8-week-old mice were used in the experiments. To generate xenografts, 106 tumor cells in 0.1 ml of serum-free DMEM were injected subcuntaneously into the flanks of the mice. Three weeks after inoculation, the mice were sacrificed, tumors were explanted and tumor sizes were determined. One half of each tumor was stored in 2% formaldehyde and embedded in paraffin for histological and immunohistochemical examination. The other half was snap frozen in liquid nitrogen for RNA and protein isolation. Results Overexpression of Gal-3 in pancreatic cancer In previous high-content microarray analyses, we have identified Gal-3 as one of the genes overexpressed in microdis- sected pancreatic cancer tissues [28]. In order to validate these results, we performed quantitative RealTime PCR (qRT-PCR) on a set of tissue samples including 10 pancreatic cancer, 5 chronic pancreatitis and 5 normal pancreatic tissue specimens. Gal-3 mRNA levels were slightly elevated in the chronic pancreatits samples, and were strongly upregulated in the majority of cancer samples (Fig. 1A), confirming the microarray data. Subsequent analysis of pancreatic cancer cell lines demonstrated strong PLoS ONE | www.plosone.org 5 June 2011 | Volume 6 | Issue 6 | e20859