ALCAM in Melanoma Motility and Adhesion While the tracking of the wound front is fairly linear in MUM-2B, the cell front of MUM-2C advances in a stop-and-start manner, due to loosely associated single cells progressing into the gap. Error bars are mean 6 S.E.M. doi:10.1371/journal.pone.0039330.g001 MUM-2C (data not shown). We next sought to determine how silencing ALCAM impacts invasive capacity of MUM-2B uveal melanoma cells. To accomplish this, we used a commercial transwell assay (CytoSelect, Cell Biolabs, Inc.) comprising an upper chamber separated from a lower chamber by a basement membrane matrix-coated 8 mm (pore size) filter. A defined number of cells were placed in the upper chamber and the cultures incubated for 8 hours, following which the number of cells that had invaded the matrix and reached the underside of the filter was counted. As expected, the sh6 and sh5rxd cell lines did not exhibit any statistically significant difference in invasive capacity compared to MUM-2B (Fig. 3B). In contrast, ALCAM- silenced sh5 cells showed a 50% reduction in invasive capacity, consistent with the similar magnitude reduction in motility observed in the gap closure assay (Fig. 3B). Figure 2. ALCAM expression is silenced in MUM-2B cells via shRNA, and rescued by re-expression of ALCAM-GFP. Western blots of MUM-2B, ALCAM-silenced sh5, control sh6, and negative control MUM-2C reveal that ALCAM protein expression is nearly completely silenced in sh5, but not in sh6 (A). Tubulin is shown as a loading control. ALCAM expression in the rescue cell line (sh5rxd, containing an ALCAM-GFP construct with point mutations to evade knockdown) is similar to MUM-2B, as shown by western blot using an anti-ALCAM antibody (B; note that the GFP tag adds ,27 kDa to the size of the protein; reprobing the blot with anti-GFP antibody detects only the rescue band, as expected). Panel (C) shows DAPI- stained nuclei of the sh5 cell line; immunostaining of sh5 confirms nearly complete knockdown of ALCAM protein expression (D, E). Panel (F) shows DAPI-stained sh6 nuclei; ALCAM expression in this cell line is robust, and localized to points of cell contact (G, H). Panel (I) shows DAPI-stained nuclei of the sh5rxd rescue cell line; ALCAM expression and localization in the rescue cells (J, K) is comparable to the sh6 cell line. Scale bar in (K) is 25 microns. doi:10.1371/journal.pone.0039330.g002 PLoS ONE | www.plosone.org 6 June 2012 | Volume 7 | Issue 6 | e39330