the means to infiltrate perivascular spaces and remodel the tumor cell surrounding provisional matrices during melanoma progres- sion. Materials and Methods Antibodies and reagents The following antibodies were used: rabbit polyclonal antibod- ies against MT1-MMP cytoplasmic tail [35] or hinge domain (RP1, Triple Point Biologics; Ab6004, Chemicon), mouse monoclonal antibodies against MT1-MMP catalytic domain (LEM2-15/8; Millipore) and hemagglutinin (HA; Covance). Rabbit polyclonal antibodies against MT3-MMP hinge domain were from Abcam (ab38968), or kindly provided by Dr. R. Fridman (Wayne State University, Detroit, USA; Ab318). The MMP inhibitor, GM6001 (Calbiochem), phalloidin-TRITC, collagen type I from rat tail (Sigma-Aldrich), bafilomycin A1 (Calbiochem) and recombinant human HGF (R&D systems) were also used. Cell culture Human Bowes melanoma cell line, derived from radial growth phase superficially spreading melanoma [30], was obtained from Dr D. B. Rifkin (Rockefeller University, New York, USA). The other human melanoma cell lines used have been established at the Wistar Institute (Philadephia, PA; [29]). They were originally derived from primary superficially spreading melanoma (G361 and WM793), nodular melanoma metastases (WM852 and WM164), or metastasis of superficially spreading melanoma (WM165 and WM239; [31,54,55]). Melanoma cells were cultivated in Eagle’s Minimal Essential Medium (MEM) contain- ing 10% heat-inactivated fetal calf serum (FCS). COS-1 monkey kidney cells were cultured according to the instructions (American Type Culture Collection). To generate stable cell pools, transfect- ed Bowes cells were selected using G418 (400 mg/ml; Calbio- chem). RNA isolation from human tissues and cultured cells Total mRNAs were extracted from representative microdissect- ed samples of archival paraffin-embedded biopsies of benign intradermal or compound melanocytic nevi (n = 11) and of adjacent normal skin (n = 2) using High Pure RNA Paraffin Kit (Roche) or from cultured cells using RNeasy Mini kit (Qiagen). The nevus biopsies had been removed for diagnostic purposes and the histological diagnosis had been confirmed by the university hospital dermatopathologist. For RNA isolation from 3D cultures, collagen and fibrin were first digested using collagenase type 2 (Worthington) and trypsin (Sigma-Aldrich), respectively (both 2 mg/ml). The cells were then collected by brief centrifugation and lysed for RNA isolation using the RNeasy Mini kit. Reverse transcription was carried out with Random hexamer primers (Invitrogen) and Superscript III reverse transcriptase (Life Technologies). In addition, TissueScan Melanoma Tissue qPCR Arrays I and II (OriGene Technologies) containing cDNAs from human tissue biopsies of normal skin (n = 6) and melanoma metastases (n = 77) were used for the quantification of the MT- MMP transcripts. qPCR The cDNAs were amplified on GeneAmp 7500 Sequence Detector thermal cycler (Applied Biosystems) using TaqMan Universal PCR Master Mix and validated primers (MT1-MMP; Hs 01037006_gH, MT2-MMP; Hs 00233997_m1, MT3-MMP; MT3-MMP in Matrix-Dependent Melanoma Invasion Hs 00234676_m1, Applied Biosystems). The expression levels were normalized with mRNA for TATA-binding protein (TBP). Cell invasion and growth in collagen Cell invasion was assessed essentially as described [33,56]. Briefly, type I collagen (4.8 mg/ml, rat tail) was mixed with equal amount of 26MEM, and pH was adjusted to ,7.4 by NaOH. Collagen was cast in the upper chambers of Falcon cell culture inserts in 24 well plates and incubated at 37uC for 1 h to allow gelling. Cross-linked fibrin gels were cast in the inserts by combining 75 ml plasminogen-free human fibrinogen (6 mg/ml; Calbiochem) in Hank’s Balanced Salt Solution (HBSS) and 75 ml HBSS (pH 7.4) containing 4 U/ml human thrombin and 400 mg/ ml aprotinin (both from Sigma-Aldrich). Cells were seeded atop the matrix in the upper chamber in medium containing 1% FCS. GM6001 (10 mM) was added to both upper and lower chambers when used. HGF (25 ng/ml) and medium containing 10% FCS served as chemoattractants in the lower chambers, with medium changed every third day. After a 5–7 d culture period, the cells were fixed with 4% PFA, and paraffin sections were stained with hematoxylin and eosin (H&E-staining). Sections were photo- graphed, and the invaded cells were counted from 8 random sections of each sample. Each assay was performed in triplicate. For 3D growth/invasion assays, collagen (2.4 mg/ml) and fibrin were prepared as above. 5000 cells were suspended in 40 ml hydrogel, the suspension were transferred to a 24-well plate, and incubated for 1 h at 37uC to allow complete gelling. After 4–16 d incubation in complete growth medium, cultures were fixed and photographed using Axiovert 200 microscope (Carl Zeiss). Alternatively, collagen or fibrin gels were stained with phalloi- din-TRITC to visualize filamentous actin, and photographed using Axioplan upright epifluorescence microscope (Carl Zeiss). The invasive areas were calculated from 20 random phase- contrast or epifluorescence images of 6 separate gels (49–163 colonies/transfected cell line, n = 3) using ImageJ software. Northern hybridization Total cellular RNA (10 mg) was fractioned by formaldehyde- agarose gel electrophoresis and transferred to a Zeta probe membrane (Bio-Rad) in 206 SSC (16 SSC: 150 mM NaCl, 15 mM sodium citrate, pH 7.0) and fixed by UV crosslinking. cDNA probes corresponding to full-length MT1-MMP and MT3- MMP cDNA were labeled with [32P] dCTP by the random priming method (Amersham Pharmacia Biotech). The membranes were hybridized in ExpressHyb hybridization solution (Clonetech) at 58uC for 16–24 h. Washing was carried out in 0.26 SSC containing 0.1% SDS at 63uC. RNA for glyceraldehyde-3- phosphate dehydrogenase (GAPDH) was used as an internal standard. RNA interference and cDNAs Small interfering RNAs (siRNA) targeting MT1-MMP (59CAGCGATGAAGTCTTCACTTA93 and 59TGGCGGGT- GAGGAATAACCAA93), MT3-MMP (59CCGCCACATACTG- TACTGTAA93 and 59AAGCACATCACTTACAGTATA93) and non-silencing control siRNAs (Qiagen) were transfected using LipofectamineTM 2000 (Life Technologies). Short hairpin RNA (shRNA) targeting MT3-MMP (shMT3-1, TRCN0000052249; shMT3-2, TRCN0000052250; and shMT3-3, TRCN0000052251) or nontargeting scrambled shRNA (Open Biosystems) in pLKO.1 vector were delivered into the cells using lentiviral transduction as described [57]. For stable shRNA expression cells were selected using puromycin (5 mg/ml). The knockdown efficiency was assessed by quantitative PCR (qPCR) after 48 h. Expression vectors for PLoS ONE | www.plosone.org 10 December 2011 | Volume 6 | Issue 12 | e28325