ALCAM in Melanoma Motility and Adhesion Figure 5. ALCAM-silenced cells display reduced MMP-2 activity. Levels of pro-MMP-2 were assayed in media conditioned by each cell line by gelatin zymography (A). Clear bands indicating pro-MMP-2 activity (which is activated by SDS; a faint ‘‘active’’ cleaved MMP2 band was present in some gels but often too weak for robust quantification) are present in MUM-2B, sh6, and the sh5rxd cell lines, but are reduced in the ALCAM-silenced sh5 cell line. MUM-2C and 2C-ALC display pro-MMP-2 levels that are lower than MUM-2B, and comparable to sh5 cells; the overexpression of ALCAM in 2C-ALC fails to increase pro-MMP-2 activity beyond that of MUM-2C. A minimum of three independent gelatin zymography trials (except 2C-ALC, 2 trials) are quantified in (B). (C) Western blots of cell lysates shows that the activation of pro-MMP-2 in the sh5 cell line is reduced (higher molecular weight band is pro-MMP-2; lower molecular weight band is active MMP-2) compared to MUM-2B, sh6, and the sh5rxd rescue cell line. doi:10.1371/journal.pone.0039330.g005 Studies describing ALCAM function in uveal melanoma, the most common form of primary intraocular cancer, are lacking. Our aim in initiating this study was to determine the role that ALCAM plays in modulating invasiveness and motility in uveal melanoma, and to provide mechanistic data that will contribute to an understanding of why ALCAM up- and down-regulation might be associated with different stages of different cancers. We describe a correlation between ALCAM expression and motility in a gap closure assay in uveal melanoma cells. These data suggested that ALCAM plays a role in promoting motility and migration. We further find that silencing of ALCAM in the invasive MUM-2B line results in decreased motility, invasiveness, and MMP-2 activation. PLoS ONE | www.plosone.org 10 June 2012 | Volume 7 | Issue 6 | e39330