primers: Forward- TGC TGG AAA CTA TGT TTG TGA GAC TG; Reverse – CTC CTG CAG AGC AGT CTC ACA AAC AT. The resulting plasmid, pEGFPN1-ALCAM-mut3 was sub- jected to PCR using primers containing BamHI and EcoRI ends to yield a full-length ALCAM fragment tagged at the C-terminus with GFP. This fragment was cloned into the pLXIN vector to yield pLXIN-ALCAM-GFP-mut3. pCMV-VSV-G, encoding a viral envelope protein was the kind gift of Chris Stipp (University of Iowa, Iowa City, IA, USA). The HEK-sh0 and HEK-sh2 cell lines were created by transient transfection of HEK cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) with either a negative control (sh0) construct in pGeneClip-hMGFP, or an shRNA against ALCAM (sh2) in pGeneClip-hMGFP (SABiosciences, Valencia, CA, USA). The sequence of the sh0 hairpin is: 59 – TCT CGG AAT CTC ATT CGA TGC ATA CCT TCC TGT CAG TAT GCA TCG AAT GAG ATT CCC T –39. The sequence of the sh2 hairpin is: 59 – TCT CGT CAG GAT GCT GGA AAC TAT GTC TTC CTG TCA ACA TAG TTT CCA GCA TCC TGA CT –39. Retroviral Transduction The stable cell lines sh5, sh6, 2C-ALC, and sh5rxd were created by retroviral transduction. The appropriate constructs were cotransfected, along with pCMV-VSV-G, into the GP2-293 packaging cell line. Conditioned media was harvested from the GP2-293 cells, filtered, supplemented with 4 mg/ml polybrene, and added to the appropriate parental cell line. The following day, the media was removed from the parental cell line, and conditioned media from the GP2-293 cells was added again. This process was repeated a total of three times. After the final day of incubation in conditioned media, the parental cells were grown for 24 hours in standard growth medium, and then appropriate selection agents were added. Gap Closure Assay/time-lapse Imaging Confluent cultures were grown in 6 cm dishes. A 10 ml pipet tip was used to inscribe a ‘‘wound gap’’ in the confluent layer of cells. Cells were washed twice with 16PBS, then RPMI-1640 media containing 10% FBS and 25 mM HEPES was added. The dishes were covered and placed on a custom stage heater that maintained media temperature at ,37 degrees Celsius. To analyze cell motility, phase contrast time-lapse microscopy was done using a QICAM camera (Q Imaging, Surrey, British Columbia, Canada) and 106 objective on a Leica DM-IRB microscope. Images were collected every 10 minutes for 8 hours, or until cells closed the wound gap. Analysis of cell migration was conducted by using QImagePro (Q Imaging, Surrey, British Columbia, Canada). The initial width of the gap was measured at three different places along the length of the wound, and the time until the cells closed the gap was recorded for each. The three speeds were calculated and averaged together to yield a value for that trial. Each reported speed represents at least three independent trials. RNA, cDNA, and RT-PCR RNA was extracted from 10 cm dishes of confluent cells using TrizolTM reagent (Invitrogen, Carlsbad, CA, USA). RNA was subsequently reverse transcribed into cDNA using the ProtoScript FirstStrand cDNA kit (NEB, Ipswich, MA, USA). cDNA was then subjected to PCR (30–35 cycles) using primers that spanned an intron of either ALCAM, MMP-2, or GAPDH. ALCAM in Melanoma Motility and Adhesion Preparation of Cell Lysates Cells were grown until confluent in 10 cm dishes, growth media was aspirated, and cells were washed twice with 16PBS. To a 10 cm dish, 1 ml of Mild Lysis Buffer (1% NP-40, 100 mM Tris-HCl pH7.4, 300 mM NaCl, 10 mM NaF, 0.5 mM sodium orthovanadate, 1 protease inhibitor cocktail EDTA-free mini tablet per 10 ml) was added. Cells were incubated for 10 minutes on ice, then scraped into an eppendorf tube. The lysate was spun at 4 degrees Celsius in a table top microcentrifuge for 10 minutes at 16,000 g to pellet cell debris. The protein concentration of the resulting superna- tant was quantified using a BCA assay. Western Blots For all western blots, 20 micrograms of total protein was loaded per sample. For all western blots except MMP-2, lysates were run on 9% acrylamide running gels with a 4% acrylamide stacking gel. Lysates blotted for MMP-2 were run on precast 4–20% gradient gels. Gels were run at 185 volts constant. Protein was then transferred to 0.45 mm nitrocellulose using a ThermoFisher electrotransfer apparatus at 0.4 amps constant. After transfer, the nitrocellulose membrane was blocked for 1 hour at room temperature in 5% dry milk in 0.05% Tween20-TBS (for rabbit and mouse primary antibodies) or in 0.05% Tween20-TBS (for goat primary antibodies). Primary antibodies were diluted in blocking solution and incubated overnight at 4 degrees Celsius. The blots were washed 3 times in 0.05% Tween20-TBS, and secondary antibodies were diluted in 0.05% Tween20-TBS and incubated with the blot for 1 hour at room temperature. The blots were washed with 0.05% Tween20-TBS three times, then developed using West Pico reagents (ThermoFisher Scientific, Rockford, IL, USA). Gelatinase Assay Cells were grown to confluency in a 10 cm dish. Growth medium was removed and replaced with 5 ml of serum-free medium for 36 hours. The media were harvested, filtered through a 0.45 micron filter to remove cells and debris, and mixed 1:1 with non-reducing sample buffer. Samples were treated as in [40], and 10 ml loaded onto an SDS-PAGE gel that included 0.1% gelatin. Invasion Assay The CytoSelectTM 24-well Invasion Assay, Basement Mem- brane, Colorimetric format (Cell Biolabs, Inc., San Diego, CA, USA) was used according to manufacturer’s protocol. Briefly, 300 ml of a 0.56106 cells per milliliter solution were plated in serum-free RPMI-1640 in the upper chamber of an insert. The bottom portion of the well was filled with 500 ml of RPMI-1640 containing 10% FBS and penicillin-streptomycin. Cells were incubated for 8 hours at 37 degrees Celsius, 5% carbon dioxide. After incubation, inserts containing invasive cells were removed and stained with the provided cell stain solution. Stained cells in three non-overlapping fields centered at the highest cell density were counted at 106 magnification on an inverted microscope. Survival Assay Cells were trypsinized, counted on a hemocytometer, and 120,000 cells were plated in triplicate in single wells of a 24-well plate. The cells were incubated for 8 hours at 37 degrees Celsius and 5% carbon dioxide, then trypsinized and counted on a hemocytometer. PLoS ONE | www.plosone.org 3 June 2012 | Volume 7 | Issue 6 | e39330