[31], bladder cancer [32], and esophageal squamous cell cancer [33], among others (for review see ref. [5]). Although ALCAM has been implicated in these numerous pathological states, it is as yet unclear how ALCAM contributes to metastasis. Existing reports are paradoxical, with ALCAM gene expression being highly upregulated in some cancers [25,31,34] and greatly downregulat- ed in others [28,35,36]. Unfortunately, these data are necessarily correlative in nature; therefore, an understanding of the contri- bution of ALCAM to cancer progression and, indeed, normal cell motility and adhesion, has been hampered by a lack of studies aimed at directly manipulating ALCAM levels within particular cell lines and determining the outcome of this manipulation. Here, we sought to address this by utilizing a number of defined uveal melanoma cell lines with high or low ALCAM levels, and testing the effects of manipulating these levels on cell motility, invasiveness, and adhesion using multiple measures. Uveal melanoma, the most common form of primary intraocular cancer, is often derived from the choroid, is highly metastatic, and results in death in 50% of patients [37]. Previous microarray analysis of two uveal melanoma cell lines identified ALCAM as one of the genes most upregulated in invasive cells (line MUM-2B) compared to non-invasive cells (line MUM-2C) [38]. We find that, across several uveal melanoma cell lines, ALCAM expression positively correlates with cell motility. Silencing of ALCAM using targeted shRNAs in MUM-2B results in both impaired cell motility and reduced invasive capacity in an in vitro assay, consistent with an observed reduction in matrix metalloproteinase activation. Con- versely, forced expression of ALCAM in the normally ALCAM- negative line MUM-2C did not increase cell motility or invasiveness, demonstrating that ALCAM is necessary but not sufficient for this cell behavior. Interestingly, we also find an effect of ALCAM on cadherin-based adherens junctions. In MUM-2C cells, forced expression of ALCAM results in increased recruitment of neural (N)-cadherin and ß-catenin to cell-cell contacts. Conversely, silencing of ALCAM expression in MUM-2B disrupts the formation of adherens junctions. These data represent the first description of ALCAM function in uveal melanoma cells, indicate cooperation between ALCAM and cadherins in mediating cell adhesion, and suggest that ALCAM’s ultimate effect on metastasis might depend on the cadherin status of surrounding tissues in conjunction with the cadherin status of the tumor cells. Methods Reagents, Cell Culture Media, Antibodies DMEM, RPMI-1640, Anti-GFP (rabbit), trypsin-EDTA solu- tion, HEPES, EHS laminin and all fluorescent-tagged secondary antibodies were from Invitrogen (Carlsbad, CA, USA). Anti-N- cadherin and anti- ß-catenin mouse monoclonal antibodies were purchased from Becton Dickinson (Franklin Lakes, NJ, USA). Anti-MMP-2 rabbit polyclonal antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Sodium orthovanadate, anti-pan-cadherin, anti- ß-tubulin, and poly-L- lysine (0.01%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-GFP (mouse) and protease inhibitor cocktail EDTA-free mini tablets were from Roche (USA). Supersignal West Pico reagents, BCA assay reagents, and HRP-conjugated anti-mouse and anti-rabbit secondary antibodies were supplied by Thermo Scientific (Rockford, IL, USA). HRP-conjugated anti- goat secondary antibody was purchased from Jackson ImmunoR- esearch (West Grove, PA, USA). Fetal Bovine Serum (FBS) was purchased from Atlanta Biologicals (Lawrenceville, GA, USA). Mouse monoclonal anti-ALCAM 3A6 antibody was purchased from Millipore (Billerica, MA, USA). Goat anti-ALCAM AF656 ALCAM in Melanoma Motility and Adhesion was purchased from R&D Systems (Minneapolis, MN, USA). Anti-ALCAM HB-2 antibody was the kind gift of Solomon Ofori- Acquah (Emory University School of Medicine, GA, USA). Puromycin and G418 were supplied by InvivoGen (San Diego, CA, USA) and Research Products International Corp. (Mt. Prospect, IL, USA), respectively. Cell Culture Conditions Cell lines were grown as follows: human uveal melanoma cell lines OCM-1A, MUM-2B, MUM-2C, C918, M619, sh5, sh6, sh5rxd, and 2C-ALC were maintained in RPMI-1640 supple- mented with 10% FBS; selection was maintained in sh5 and sh6 with 1 mg/ml puromycin; in sh5rxd with 1 mg/ml puromycin and 250 mg/ml G418; in 2C-ALC with 250 mg/ml G418. HEK cells and the retroviral packaging cell line GP2-293 was maintained in DMEM supplemented with 10% FBS. All cell lines were maintained at 37 degrees Celsius in 5% carbon dioxide. The MUM-2B and MUM-2C lines were the kind gift of Dr. Elizabeth Seftor (Children’s Memorial Research Center, Chicago, IL, USA); the OCM-1A, C918, and M619 lines were the kind gift of Dr. Karla Daniels (University of Iowa, Iowa City, IA, USA). Immunohistochemistry Cells were grown in 24-well dishes on glass coverslips coated with poly-L-lysine (0.01%) and laminin (10 mg/ml). Cells were grown on coverslips until at the desired confluency, then fixed for 15 minutes in 4% paraformaldehyde, rinsed with 16PBS, and blocked in standard blocking solution (2.5% BSA, 0.1% Triton X- 100, 0.02% sodium azide) for 1 hour. Primary antibodies were diluted in blocking solution and incubated on coverslips overnight at 4 degrees Celsius. Coverslips were rinsed in 16PBS and secondary antibody diluted in 16PBS was added for 1 hour at room temperature. Coverslips were rinsed in 16PBS containing DAPI (49,6-diamidino-2-phenylindole) to stain nuclei, then rinsed once with water and set on filter paper to dry. After drying, coverslips were mounted on glass slides using GelMount (Biomeda Corporation, Foster City, CA, USA). ß-catenin-positive regions between two cells were scored as strong, weak, or non-existent as shown below in the text and figures. Contacting cells in a minimum of three independent fields were scored. Plasmids and Vectors The sh5 and sh6 cell lines were created using viral transduction of shRNA constructs in the pSIREN-RetroQ vector. Hairpin sequences were cloned into the vector between 59 BamHI and 39 EcoRI sites. The sequence of the sh5 insert is: 59-GAT CCG TAT GTC TGC GAA ACT GCT CTG TTC AAG AGA CAG AGC AGT TTC GCA GAC ATA TTT TTT CTA GAG-39. The sequence of sh6 is: 59-GAT CCG TCA AGC AAC CAT CTA AAC CTG TTC AAG AGA CAG GTT TAG ATG GTT GCT TGA TTT TTT CTA GAG-39. The shRNA-containing plasmids were cotransfected into GP2-293 cells along with pCMV- VSV-G to produce virus particles. The 2C-ALC cell line was created by viral transduction of pWD201 into MUM-2C cells. The plasmid was previously described in [39]. To create the sh5rxd cell line, sh5 cells were transduced a second time with the construct pWD-ALCAM-GFP-mut3. This plasmid was created by first cloning full-length ALCAM from pWD201 into pEGFPN1 at SalI and AgeI sites to create pEGFPN1-ALCAM. To avoid shRNA knockdown of the rescue plasmid, three silent mutations were introduced into this plasmid by use of the Stratagene QuikChange kit, using the following PLoS ONE | www.plosone.org 2 June 2012 | Volume 7 | Issue 6 | e39330