MT3-MMP, MT1-MMP, MT1-MMP with inactivating E240A point mutation (MT1E/A) and HA-tagged MT1-MMP have been described [17,18,58] and were transfected using FuGENE (Roche) or TransIT-2020 (Mirus). Inactive MT3-MMP E247A construct (MT3E/A) was kindly provided by Dr. D. Pei [34]. Rescue constructs for MT3-MMP with double silent mutations in shMT3-2 binding sequence rescMT3-1 (T822C-T825C) and rescMT3-2 (T831C-A836C) were generated with primers (59CTACAGT- GAATTAGAAAATGGCAAACGCGACGTGGATATAACC93 and 59GGTTATATCCACGTCGCGTTTGCCATTTTCTAA- TTCACTGTAT93) and (59GGCAAACGTGATGTGGACAT- CACCATTATTTTTGCATCTGG93 and 59CCAGATGCAAA- AATAATGGTGATGTCCACATCACGTTTGCC93), respec- tively, using QuikChangeH Site-Directed Mutagenesis Kit (Strata- gene). Immunoblotting Cells were lysed with RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% NaDOC, 0.1% SDS) containing CompleteH protease inhibitor cocktail (Roche). Protein contents of the lysates were determined by bicinchoninic acid protein assay (Sigma-Aldrich). Equivalent quantities of protein were size-fractionated by gradient (4–20%) SDS-PAGE followed by transfer to polyvinylidene difluoride membranes. The non-specific protein binding sites were saturated with 5% non-fat milk in TTBS-buffer (TBS containing 0.05% Tween-20) for 30 min. The membranes were washed with TTBS and incubated with primary antibodies overnight, and with biotinylated secondary antibodies (Dako) for 30 min. After incubation with horseradish peroxidase –conjugated streptavidin for 20 min (GE Healthcare), the bound antibodies were detected using enhanced chemiluminescence (Promega). All experiments were repeated for 2–4 times. Immunoprecipitation Endogenous or recombinant MT1-MMP and MT3-MMP were immunoprecipitated from cell lysates by incubation with specific rabbit polyclonal antibodies for MT3-MMP (against hinge domain; Ab38968) or MT1-MMP (against cytoplasmic tail [35] or hinge region; AB6004, Chemicon) and G protein sepharose (GE Healthcare) for 2 h at 4uC. The sepharose particles were then pelleted by centrifugation and washed extensively with PBS. The proteins were eluted with the sample buffer and separated for immunodetection by SDS-PAGE. Cell surface labeling Cell surface biotinylation was performed as described [35]. Briefly, cells were rinsed twice with PBS and incubated with 0.5 mg/ml biotin (Pierce) in PBS on ice for 1 h. The reaction was terminated by washing 3 times for 10 min with 150 mM glycine/ TBS. The cells were then lysed and subjected to immunoprecip- itation with rabbit polyclonal MT1-MMP or MT3-MMP antibodies. The immunoprecipitated material was resolved by SDS-PAGE, and detected with horseradish-peroxidase-conjugated streptavidin (Daco). Immunofluorescence The cells on glass coverslips were fixed with 4% PFA followed by staining with the indicated primary antibodies and Alexa Fluor- labeled secondary antibodies (Invitrogen). The coverslips were finally mounted on glass slides using Vectashield anti-fading reagent (Vector Laboratories) and examined using the Axioplan microscope. MT3-MMP in Matrix-Dependent Melanoma Invasion Gelatin zymography MMP-2 was transiently expressed in COS-1 cells, and the conditioned medium was harvested after 18 h. Aliquots of the conditioned medium were incubated with COS-1 cells overex- pressing MT1- and MT3-MMPs for 24 h. Aliquots of conditioned medium were then subjected to the gelatin zymography [57]. Statistical analysis All numerical values represent mean 6 SD. Statistical significance was determined using the Mann-Whitney test. Supporting Information Figure S1 MT1-MMP and MT3-MMP expression in melanoma cells cultured within 3D collagen and fibrin. Relative values of average MT1-MMP (A) and MT3-MMP (B) mRNA expression in indicated melanoma cell lines cultured inside 3D collagen and fibrin gels for 48 h were analyzed by qPCR. MT1-MMP and MT3-MMP mRNA expression in Bowes cells cultured in collagen were set to 100%, n = 3. (TIF) Figure S2 Catalytically inactive MT3-MMP does not activate MMP-2. Gelatin zymogram shows MT1-MMP-medi- ated MMP-2 activation by COS-1 cells expressing MT1-MMP, MT3-MMP or MT3E/A. MMP-2: latent (L), intermediate (I) and active (A). After harvesting of conditioned media, the cell lysates were subjected to immunoblotting for MT3-MMP and MT1- MMP. (TIF) Figure S3 MT3-MMP reduces MT1-MMP on the cell surface. COS-1 cells transiently expressing MT1-MMP or MT1- MMP in combination with MT3-MMP or MT3E/A were fixed and stained with antibodies against the hinge domain (RP1) and catalytic domain (LEM2-15/8) of MT1-MMP. Magnified areas indicated by white boxes are presented below the each image for RP1 staining, and on figure 5E for LEM2-15/8 staining. (TIF) Figure S4 MT3-MMP modulates collagen and fibrin invasion of three different melanoma cell lines. (A) Average MT1-MMP and MT3-MMP mRNA expression in the WM852 cell pools expressing shRNA against MT3-MMP (shMT3-1, shMT3-2 and shMT3-3) relative to cells expressing scrambled shRNA (Scr). (B) WM852 cells stably expressing indicated shRNAs were embedded in 3D collagen type I or fibrin gels as single cell suspension. Light micrographs show represen- tative cell colonies that were formed after 7-d assay. (C) Average MT-MMP mRNA expression in the WM165 cells expressing indicated shRNAs. (D) WM165 cells expressing indicated shRNAs were cultured inside 3D collagen type I and fibrin for 14 d, after which cultures were fixed and stained for filamentous actin. Quantifications of invasive areas are presented on figure 6B. (E) Average MT-MMP mRNA expression in the WM793 cells stably expressing indicated shRNAs. (F) Light micrographs show representative WM793 cell colonies that were formed in 3D collagen and fibrin after 16-d assay. (TIF) Figure S5 Silencing of MT3-MMP does not have any notable effect on Bowes cell invasion. (A) MT1-MMP and MT3-MMP mRNA levels in Bowes cells transfected with control siRNA (Ctrl) or siRNAs targeting MT1-MMP (siMT1) or MT3- MMP (siMT3) were detected by qPCR (n = 3). (B) Cells transfected with the indicated siRNAs were allowed to invade PLoS ONE | www.plosone.org 11 December 2011 | Volume 6 | Issue 12 | e28325