here, such as co-receptors and the ERM proteins, are often overexpressed in cancer [7,36,37,38]. Thus, our proposed model of ezrin-mediated activation of SOS now allows us to conceive how such inappropriate activation of co-receptors, e.g. mis- expression of the co-receptor CD44v6 for the RTK c-Met [39], as well as elevated expression of ezrin [37], radixin [38] and moesin [36] may contribute to cancer progression and metastasis. Methods Growth factors, antibodies and reagents Recombinant human platelet-derived growth factor BB (PDGF) (Biomol); recombinant human interleukin-6 (IL-6), epidermal growth factor (EGF), lysophosphatidic acid (LPA), 12-o-tetrade- canoyl-phorbol-13-acetate (TPA), Igepal CA-630, Triton-X-100, GDP, GTPcS and doxycycline (dox; Sigma); mantGDP, mantGTP and GST protein (Jena Bioscience); Lubrol 17A17 (Uniqema); ATP (Roche); GST-Grb2 glutathione agarose (GST- Grb2), Raf1-Ras-binding domain glutathione agarose (GST-Raf1- RBD), GST-Ras- and GST-agarose (Upstate); latrunculin B (Calbiochem); glutathione agarose (Santa Cruz). Antibodies: GST (B-14), Integrin-b1 (M-106), Erk1 (K23), Ezrin (C-19), moesin (C-15), radixin (C-15), Actin (I-19), SOS1 (C-23), PDGFR- b (958), GFP (B-2), Myc (9E10), Tubulin (TU-02) (Santa Cruz); Ras (RAS10) (Upstate); VSVg, HA (Roche); Flag (F1804) (Sigma); ezrin (3C12) (NeoMarkers); against phosphorylated proteins: PDGFR-b (Y716) (Upstate); Erk (Thr202/Tyr204), Stat3 (Tyr705), CREB (Ser133), ezrin(Thr567)/radixin(Thr564)/moesin (Thr558) (Cell Signalling). Plasmids EzrinWT-, N- and Cterminal-GFP, and ezrin(DABD)-EGFP- N1 (Antonio Sechi, Braunschweig). EzrinWT- and T567D-VSVg (pCB6), ezrin N- and Cterminal-GST, Human ezrinWT- and T567D-Myc in pcDNA3.1 (Monique Arpin, Paris). EzrinWT- and T567D-VSVg fragments were subcloned into pUHD10.3 rtTA- responsive cloning vector (EcoR1/Xba1). Ezrin Nterm- and Cterm-GST were cloned into pGEX4T-1 vector (BamH1/ EcoR1). Human moesinWT (Dominique Lallemand, Paris) expression construct was generated in pcDNA3.1 (Kpn1/Not1), and a dox-inducible radixinWT-Flag (pcDNA3 Human radix- inWT-Flag, David Gutmann, St. Louis, Washington) was created in pUHD10.3 (BamH1/EcoR1). Myc-SOS and His-SOScat constructs were generated by PCR, then cloned into the pcDNA3.1/V5-His-TOPO vector (Invitrogen). GST-CD44tail and GST-CD44tail mutant in pEBG-GST were generated as described in Morrison et al., 2001. Myc-H-Raps and HA-RasWT in pcDNA3.1 (UMR cDNA Resource Center, University of Missouri-Rolla, USA). For protein purification, ezrinWT-His was cloned in pET15TEV expression vector (C. Breithaupt-Than, Halle). His-SOScat (aa 564–1094) in pProEX HTb expression vector was obtained from A. Wittinghofer, Dortmund. His-SOS DH-PHcat (aa 198–1049), His-SOS HDPcat (aa 1–1049) and His- H-Ras (aa 1–166) were cloned in pET15TEV expression vector (BamH1). All point mutations were generated using a site-directed mutagenesis kit (Stratagene). siRNA experiments The down-regulation of ERM proteins was carried out using short interfering RNA (siRNA) according to the procedure described in [40]. Single siRNA oligonucleotides were purchased from Ambion (only the sense strand is reported): ezrin (rat, Set I): 59-UCAACUAUUUCGAGAUCAAAA-39 (ORF 606–629), ezrin (rat, Set II): 59-GGGACUCAGGCCUGUUUAUtt-39 (ORF Ras Requires ERMs 2710–2732); radixin (rat, Set I): 59-CUCGUCUGAGAAU- CAAUAAGC-39 (ORF 752–775), radixin (rat, Set II): 59- GCUGUGGCUGGGCGUUGAUtt-39 (ORF 852–874); moesin (rat, Set I): 59-GGCUGAAACUCAAUAAGAAGG-39 (ORF 171–194), moesin (rat, Set II): 59-GGCCCUGCUGCAGGCUU- CUtt-39 (ORF 1299–1321); ezrin (mouse, Set I): 59-GCCGUAU- GUAGACAAUAAAGG-39 (ORF 139–161), ezrin (mouse, Set II): 59-GGCCAAGUUCGGAGAUUAUtt-39 (ORF 528–550); ra- dixin (mouse, Set I): 59-GCACCUCGUCUGAGAAUCAAU-39 (ORF 809–822), radixin (mouse, Set II): 59-GCUGUGGCUAG- GUGUUGAUtt-39 (ORF 1195–1217); moesin (mouse, Set I): 59- GCAAGCCUGACACCAUUGAGG-39 (ORF 882–905), moesin (mouse, Set II): 59-GGCCCUGCUGCAGGCUUCUtt-39 (ORF 1388–1410); Control (Ctr) siRNA (against firefly luciferase) 59- CGUACGCGGAAUACUUCGA-39. SMART pools (cocktail of four siRNAs) against ezrin (mouse), moesin (mouse) and radixin (mouse) were purchased from Dharmacon (sequences will be provided upon request). The down-regulation of ERM proteins in HeLa and RPM-MC was carried out the following siRNA duplexes: ezrin (human, Set I): 59-CCCCAAAGAUUGG- CUUUCC-39 (ORF 704–722), ezrin (human, Set II); 59- UCCACUAUGUGGAUAAUAA-39 (ORF 140–158); moesin (human, Set I): 59-AAAAGCCCCGGACUUCGUC-39 (ORF 786–804), moesin (human, Set II): 59-AGAUCGAGGAACAGA- CUAA-39 (ORF 1058–1076); radixin (human, Set I): 59-GCA- GUUGGAAAGGGCACAA-39 (ORF 948–966), radixin (human, Set II): 59-CUCGUCUGAGAAUCAAUAA-39 (ORF 752–775). As control, the following scrambled duplexes (derived from Set I duplexes) were used: ezrin 59-CCGUCACAUCAAUUGCCGU- 39, moesin 59-ACUAGACGAACCGUCGCUC-39, radixin 59- GAUGCAGCAGCAUGAAGAG-39. Cell culture RT4-D6P2T (RT4) rat schwannoma cell line ENU-induced RT4-D6P2T rat schwannoma cell line (mutated ErbB2 activates the Ras-MAP kinase cascade) [41], NIH 3T3 mouse fibroblasts and HeLa human cervix epitheloid carcinoma cell line (European Collection of Animal Cell Cultures). RT4 and NIH 3T3 cells carrying doxycycline (dox)-inducible ezrin wild type or ezrin mutants (named either RT4tetEzrin or NIH 3T3tetEzrin) were prepared as described in Morrison et al., 2001. Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; Gibco-BRL) supplemented with 10% foetal calf serum (FCS; Gibco-BRL) or 10% donor calf serum (Gibco-BRL) for NIH 3T3. All cells were maintained in a humidified atmosphere with 5% CO2 at 37uC. The concentration of dox in all cell culture experiments was 1 mg/ml. Stable and transient transfection of cells Transfections (both RNAi and plasmid DNA) were performed using Lipofectamine (Invitrogen), in accordance with the manu- facturer’s instructions. To generate stable or pooled clones, cells were co-transfected with pCEP4 (hygromycin resistance) (Invitro- gen), and selected in 100 mg/ml hygromycin (Roche). Note, dox- inducible ezrin wild type or ezrin mutants as well as ezrin expressing stable/pooled clones over-expressed exogenous ezrin more than 35 times compared to endogenous ezrin levels. Definition of growth condition in culture dishes Low cell density ( = logarithmic or exponential growth) was defined as the density recorded at 24 h after seeding 500 cells per cm2. High cell density ( = confluent growth condition) was defined as the density recorded at 24 h after seeding 56103 cells per cm2 (16105 cells per cm2 for NIH 3T3). PLoS ONE | www.plosone.org 10 November 2011 | Volume 6 | Issue 11 | e27511