Figure 5. No effect of transient Gal-3 knockdown on caspase- dependent apoptosis in PaTu 8988s cells. Cells transiently transfected with Gal-3 specific siRNAs (siGal-3 1 and 2), nonsilencing control siRNA (NC) or untransfected cells (U) were analyzed by Western blot for poly ADP-ribose polymerase (PARP) cleavage. Increased cleavage, indicated by the enhanced signal of the lower band, was seen in all transfected cells. PARP cleavage was enhanced by treatment with the extrinsic apoptosis inducer Trail, but was not influenced by Gal-3 knockdown. b-actin was used as loading control. Results shown are representative of three individual experiments. doi:10.1371/journal.pone.0020859.g005 expression, both on the mRNA as well as on the protein level, in 5 out of 6 tested cell lines (Fig. 1B). Expression level of Gal-3 has no influence on tumor cell growth in vitro To assess the functional role of the aberrantly expressed Gal-3 in pancreatic cancer cells, Gal-3 was transiently or stably overexpressed or silenced in different pancreatic cancer cell lines and the effects analyzed in various in vitro functional assays. For transient silencing, two independent siRNA sequences as well as a non-silencing control siRNA were used. For each of the five cell lines analyzed (PaTu 8988s, S2-007, S2-028, IMIM-PC-1 and MIA PaCa-2), transfection reagents and –conditions were optimized to achieve knockdown efficiencies .80% (Fig. 2A). Stable knockdown clones were generated using S2-007 cells transfected with shRNA sequences cloned in the pGIPZ vector. Three stably transfected clones as well as three control vector transfected clones were chosen for further analysis (Fig. 2B). Since PaTu 8988t cells showed very low endogenous Gal-3 levels, this cell line was chosen for the construction of stable Gal-3 overexpressing clones. Three independent clones were established. Two of these showed moderate overexpression of recombinant Gal- 3 on the mRNA level, which was much more pronounced on the protein level (Fig. 2C, lanes 4, 6). The remaining clone did not show appreciable expression of recombinant Gal-3 (Fig. 2C, lane 5), but was included in the further experiments as additional control clone. Interestingly, control clones transfected with empty vector also showed somewhat elevated Gal-3 levels (Fig. 2C, lanes 2, 3). In a first functional assay, the influence of Gal-3 expression on cell viability was analyzed by MTT assays. Neither transient knockdown in any of the 5 tested cell lines, nor stable knockdown in S2-007 cells or stable overexpression in PaTu 8988t cells had any significant effect on cell viability under normal culture conditions as compared to the suitable controls (Fig. 3 A–C). Analysis of cell proliferation using BrdU assays revealed a moderate, but significant inhibition of proliferative activity in S2- 028 cells after knockdown of Gal-3 (Fig. 4A, panel 3). However, the trend to reduced proliferation did not reach significance in Galectin-3 in Pancreatic Cancer PaTu 8988s and S2-007 cells, was absent in IMIM-PC-1 and even reversed in MIA PaCa-2 cells (Fig. 4A). Neither stable knockdown in S2-007 cells, nor stable overexpression of Gal-3 in PaTu 8988t cells had any significant effect on proliferation of the resulting cell clones. Likewise, transient Gal-3 knockdown had no effect on apoptotic activity (as measured by PARP cleavage) of PaTu 8988s cells in the absence (Fig. 5, lanes 1–4) or presence (Fig. 5, lanes 5–8) of the extrinsic apoptosis inducer Trail. PARP cleavage was induced by the transfection procedure and was more pronounced in the presence of Trail (Fig. 5, lanes 2 and 6), but was not further enhanced by knockdown of Gal-3 (Fig. 5, lanes 3–4 and 7–8). Replication of this set of experiments under serum-free conditions produced similar results, failing to demonstrate any consistent influence of the modulation of Gal-3 expression on cell growth or apoptosis in the tested cell lines (data not shown). Inconsistent effect of Gal-3 on cell migration We ext analyzed the influence of Gal-3 expression on cell migration in a Boyden chamber assay. Cells were able to transmigrate through a filter along a fetal calf serum gradient. Numbers of transiently Gal-3 silenced migrated cells were normalized to non-silencing control siRNA transfected cells. Transient knockdown of Gal-3 resulted in a trend towards decreased cell migration in three out of five analysed cell lines, although this effect did not reach significance in most instances (Fig. 6A). Moreover, this effect was not reproduced in the S2-007 cell clones with stable Gal-3 knockdown (Fig. 6, B). In reverse analogy to the results of the transient knockdown experiments, stable overexpression of Gal-3 in PaTu 8988t cells induced a trend towards increased cell migration (Fig. 6, C), although this trend again did not reach significance and was also apparent in clone pGal-3 2, which showed low or absent expression of recombinant Gal-3 (see Fig. 2C). Taken together, modulation of Gal-3 expression had a mild and rather inconsistent effect in the Boyden chamber assays, thus arguing against an important role of cellular Gal-3 in directed migration of pancreatic cancer cells. Inhibition of Gal-3 expression impairs anchorage independent growth in a subset of pancreatic cancer cell lines but has no effect on growth of xenograft tumors in vivo The potential for anchorage-independent growth of the cancer cells was assessed by evaluating the numbers of colonies formed in soft agar assays. Transient knockdown of Gal-3 resulted in a clear reduction in colony formation capacity for the cell lines PaTu 8988s, S2-007 and IMIM-PC-1, with the strongest effect and highest statistical significance observed for S2-007 cells (Fig. 7A). Conversely, S2-028 and MIA PaCa-2 cells were not affected by the Gal-3 knockdown. Since previous reports had indicated that tumor-promoting functions of Gal-3 in other cell systems may depend on the subcellular localization of the protein [26,36,37], we analyzed whether the differential effect of Gal-3 knockdown colony forming capacity correlated with different subcellular localization of Gal-3 in the tested cell lines. Gal-3 protein was evenly distributed between nucleus and cytosol in MIA PaCa-2 and S2-007 cells and was slightly overrepresented in the cytosolic fractions in PaTu 8988s, S2-028 and IMIM-PC-1 cells (Fig. 7B), thus showing no correlation with sensitivity towards Gal-3 knockdown. We then tested if the effect on anchorage-independent growth in S2-007 cells also translated into differences in tumor growth in vivo. PLoS ONE | www.plosone.org 6 June 2011 | Volume 6 | Issue 6 | e20859