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Supplementary Materials and Methods Cell lines and Antibodies. SW480 and HCT116 human colon carcinoma cell lines, CT26 murine colon carcinoma cell line were purchased from ATCC. Cos-7 cells were from ATCC. The following antibodies were used: monoclonal antibody anti-CD81 (clone1.3.3.22 , Santa Cruz Biotechnology), monoclonal antibody anti-ADAM10 ectodomain (clone 163003, R&D systems), goat polyclonal antibody anti-ADAM10 (A-20, Santa Cruz Biotechnology), rabbit polyclonal antibody anti-RhoA (119, Santa Cruz Biotechnology), rabbit polyclonal antibody anti-GFP (FL, Santa Cruz Biotechnology), rabbit polyclonal antibody anti-Gα13 (A-20, Santa Cruz Biotechnology), rabbit polyclonal antibody anti-Phospho-Ezrin (Thr567)/Radixin(Thr564)/Moesin(Thr558) (#3141, Cell Signaling Technology), rabbit polyclonal antibody anti-Phospho-Myosin Light Chain2 (Ser19)(#3671, Cell Signaling Technology), monoclonal antibody anti-CD9, clone FMC56 was provided by Dr. H. Zola, monoclonal antibody anti-α-tubulin (clone DM1A, Sigma). Shotgun Proteomics. The application of this technique is the analysis of protein complexes isolated by immunoprecipitation to identify protein interactions and binding partners. This method replaces the conventional gel-based methods with bi-dimensional liquid chromatography that is more sensitive in revealing peptides present in low concentrations. To immunoprecipitate CD81- associated proteins, anti-CD81 antibody 1.3.3.22 was coupled to protein A Sepharose beads and incubated with the pre-cleared lysate of SW480 overnight at 4°C. The precipitated proteins were resuspended in 8M urea and 100mM Tris, pH 8.5. Proteins were digested and Multidimensional Protein Identification Technology (MudPIT) was carried out as described with a bi-dimensional LC-ESI-MS/MS on a Finningan LQC ion trap instrument [Link et al 1999]. Using the SEQUEST algorithm, collected MS/MS spectra of peptides were correlated with predicted amino acid sequences in translated genomic databases. The resulting list of peptide sequences identified the proteins in the starting complex. Gelatinolytic Zymography. SW480 cell lysates and conditioned media from the SW480 cultures were immunoprecipitated with antibodies anti-ADAM10 or anti-CD81 and analyzed for gelatin degradation activity by SDS-PAGE under nonreducing conditions. The gel contained 5.8 mg/ml gelatin and 8% acrylamide. After one wash with water, the SDS was extracted by incubating the acrylamide gel in a 2% Triton X-100/PBS solution. The gel was then incubated in a buffer containing 5 mM CaCl2, 150 mM NaCl, and 50 mM Tris at 37 °C for 16 h and the gelatinolytic activities were visualized by staining the gel with Coomassie Blue R-250. Expression of ADAM10 truncation mutants. The plasmid pcDNA3-ADAM10 coding for wild-type bovine ADAM10 (GenBank: Z21961.1) was used as template to clone ADAM10 into pEGFP-N1 (Clontech) or pTagRFP-N1 (Evrogen) plasmids and construction of truncation mutants of ADAM10 by PCR using XhoI and KpnI restriction enzymes. Disintegrin and cysteine-rich domain ADAM10 (DisCy-EGFP 420-721) Forward primer sequence: 5’CGCCTCGAGATGGCAAGAGCAACATCT3’. Reverse primer sequence: 5’CGCGGTACCACACGTCTCATATGTCCCAT3’. Cysteine-rich domain ADAM10 (Cys-EGFP 554-721) Forward primer sequence:5’CGCCTCGAGATGGACTGTAATAGACAT3’ Reverse primer sequence: 5’CGCGGTACCACACGTCTCATATGTCCCAT3’ Transmembrane and intracytoplasmic domain ADAM10 ( TMCyt-EGFP 677-721) Forward primer sequence:5’ CGCCTCGAGATGTACTGGTGGGCAGTA3’ Reverse primer sequence: 5’CGCGGTACCACACGTCTCATATGTCCCAT3’ Mutants were transfected into Cos-7 cells using Arrest-In transfection reagent (Open Biosystems, ThermoFisher scientific). RhoA activity assay. SW480 and CT26 were plated onto control dishes (100mm) or dishes coated with supernatant of Cos-7 transfected with ADAM10Cys-EGFP or ADAM10-EGFP 60 min. Cells were lysed in 0.5 ml lysis buffer (50 mM Tris, pH 7.4, 10 mM MgCl2, 500 mM NaCl, 1% Triton X-100, 10 µg/ml each of aprotinin and leupeptin, 1 mM phenylmethylsulfonyl fluoride, and 200 μM sodium orthovanadate) and lysates were cleared by centrifugation at 15,000 g for 10 minutes at 4°C. The supernatants were then incubated for 1 hour with 30 μg purified GST-Rhotekin RhoA-binding domain fusion protein (GST-RBD) bound to the glutathione-Sepharose 4B beads, washed three times with washing buffer (50mM Tris, pH 7.4, 10 mM MgCl2, 150 mM NaCl, 1% Triton X-100) and then immunoblotted with an anti-RhoA polyclonal antibody. An anti-RhoA antibody was used as loading control. RNA interference. Two different Gα13 siRNA target sequences were used: siRNA sequence for murine Gα13 , 5’-GTCCACCTTCCTGAAGCAG3’. siRNA sequence for human Gα13, 5’GGATAAACTTGGAGAACCA3’. Scrambled siRNA sequence was : 5'GAGGAGCCGACGCTTAATA-3'. The siRNAs were chemically synthesized by Qiagen. For transfection, the Amaxa nucleofection technology™ (Amaxa) was employed as previously described (Mazzocca et al 2008). 72 h after transfection, the protein expression levels were analyzed by immunoblotting or immunofluorescence. The efficiency of transfection was at least 80% as detected by Alexa Fluor 488-labelled control siRNA. Retroviral infections. We cloned by PCR the EcoRI and SalI-flanked insert from plasmid pRetroQ ADAM10-GFP containing ADAM10 Cys rich domain-GFP cDNA into retroviral vector pBabepuro (Addgene). Furthermore, we cloned the EcoRI and XhoI–flanked fragment from vector pRetroQ-DsRedmonomer (Clontech), containing the full-length monomeric RFP cDNA, into retroviral vector pLXSN (Clontech). Retroviral particles were generated by transient transfection of the Phoenix-amphotropic packaging cell line (Allele Biotech). Phoenix cells plated onto 100mm dishes were transfected at approximately 80% confluence with 20 µg of plasmid using Arrest-In transfection reagent. After 48 h, virus-containing supernatant was recovered centrifuged and used immediately or aliquoted and frozen at -80°C. Target cells were infected with virus-containing supernatant in the presence of polybrene (8µg/ml, Sigma). We made pBabeN19RhoA-AcGFP1 subcloning the BamHI-EcoRI fragment from plasmid pCEV29N19RhoA into BamHI-EcoRI sites of retroviral vector pBabe-puro, successively we cloned by PCR the HindIII and ClaI-flanked insert from pRetroQ-AcGFP1 (Clontech) containing AcGFP1cDNA into pBabe-puro replacing the puromycin cDNA. Retroviral particles were generated by transient transfection of the Phoenix-amphotropic packaging cell line. Gene Knockdown assay The following shRNAs (OriGene Technologies) were stably delivered into CT26 and HCT116 by retroviral infection according to manufacturer’s instructions: TG509819, is a plasmid containing 29 mer shRNA construct in retroviral GFP vector to knockdown mouse CD9 ; TG314060 is a plasmid containing 29 mer shRNA construct to silence human CD9; TR30013 is a non-effective 29 mer scrambled shRNA used as control. Retrovirally transduced CT26 and HCT 116 cells were selected in puromycin-containing medium and, after 1–2 weeks, reduced expression of CD9 protein and mRNA was assessed by flow cytometry and RT-PCR, respectively. Next, silenced CT26 were stably transfected with EGFP (106 cells) and mixed with silenced CT26 stably transfected with RFP (106 cells) in the presence of geneticin. The same procedure was used with HCT116 cells. Fused cells were identified by dual color FACScan.