soluble/cytosol (supernatant) fraction. Membrane preparations were resuspended in a reaction mixture of 1 mM MnCl2, 20 mM Hepes pH 7.4, 5 mg/ml BSA, 5 mM ATP, and incubated, or not, for 45 min. with 1 mM AG1478 at 4uC, prior to the addition (or not) of either 100 ng/ml EGF or 300 mM H2O2 (stock solution in water from Sigma). The reaction was carried out at 37uC for 20 min. and stopped by the addition of the SDS-PAGE loading dye. Alternatively, the cells were scraped directly in 1 mM MnCl2, 20 mM Hepes pH 7.4, 5 mg/ml BSA, 5 mM ATP and homoge- nized by several passages in a 30 gauge needle; unbroken cells and nuclei were removed by centrifugation for 5 min. at 5006g and the supernatant, containing the broken cells, was used directly for the in vitro kinase assay (without 60,0006g centrifugation step). Measurement of cholesterol levels Cell cholesterol levels were measured by using a quantitative assay from Cell Technology, following manufacturer instructions. Briefly, after the treatments, A549 cells were washed, scraped in PBS and divided in two aliquots. One aliquot was used to determine total protein amount (normalization step) and to test the treatments. The other aliquot was used to extract total lipids and measure cholesterol levels. Lipids were extracted with chloroform: methanol (2: 1) then mixed with 1 M NaCl, prior to phase separation via centrifugation (5 min. at 15006g). Lipids in the organic phase were collected, dried under nitrogen and resus- pended in 0.1 M KPO4, 50 mM NaCl, 5 mM deoxycholic acid, 0.1% Triton-x 100. Aliquots of the resuspended lipids were measured for cholesterol content in the reaction mixture (Cell Technology protocol). References 1. Yarden Y, Sliwkowski MX (2001) Untangling the ErbB signalling network. Nat Rev Mol Cell Biol 2: 127–137. 2. Lu Z, Jiang G, Blume-Jensen P, Hunter T (2001) Epidermal growth factor- induced tumor cell invasion and metastasis initiated by dephosphorylation and downregulation of focal adhesion kinase. Mol Cell Biol 21: 4016–4031. 3. Schlessinger J (2002) Ligand-induced, receptor-mediated dimerization and activation of EGF receptor. Cell 110: 669–672. 4. Schlessinger J (1988) The epidermal growth factor receptor as a multifunctional allosteric protein. Biochemistry 27: 3119–3123. 5. Cochet C, Kashles O, Chambaz EM, Borrello I, King CR, et al. (1988) Demonstration of epidermal growth factor-induced receptor dimerization in living cells using a chemical covalent cross-linking agent. J Biol Chem 263: 3290–3295. 6. Schlessinger J (2000) Cell signaling by receptor tyrosine kinases. Cell 103: 211–225. 7. Hynes NE, Lane HA (2005) ERBB receptors and cancer: the complexity of targeted inhibitors. Nat Rev Cancer 5: 341–354. 8. Ogiso H, Ishitani R, Nureki O, Fukai S, Yamanaka M, et al. (2002) Crystal structure of the complex of human epidermal growth factor and receptor extracellular domains. Cell 110: 775–787. 9. Garrett TP, McKern NM, Lou M, Elleman TC, Adams TE, et al. (2002) Crystal structure of a truncated epidermal growth factor receptor extracellular domain bound to transforming growth factor alpha. Cell 110: 763–773. 10. Dawson JP, Berger MB, Lin CC, Schlessinger J, Lemmon MA, et al. (2005) Epidermal growth factor receptor dimerization and activation require ligand- induced conformational changes in the dimer interface. Mol Cell Biol 25: 7734–7742. 11. Red Brewer M, Choi SH, Alvarado D, Moravcevic K, Pozzi A, et al. (2009) The juxtamembrane region of the EGF receptor functions as an activation domain. Mol Cell 34: 641–651. 12. Zhou W, Ercan D, Chen L, Yun CH, Li D, et al. (2009) Novel mutant-selective EGFR kinase inhibitors against EGFR T790M. Nature 462: 1070–1074. 13. Kumar A, Petri ET, Halmos B, Boggon TJ (2008) Structure and clinical relevance of the epidermal growth factor receptor in human cancer. J Clin Oncol 26: 1742–1751. 14. Yun CH, Boggon TJ, Li Y, Woo MS, Greulich H, et al. (2007) Structures of lung cancer-derived EGFR mutants and inhibitor complexes: mechanism of activation and insights into differential inhibitor sensitivity. Cancer Cell 11: 217–227. 15. Paez JG, Janne PA, Lee JC, Tracy S, Greulich H, et al. (2004) EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 304: 1497–1500. EGFR Activation under Oxidative Stress Immuno-fluorescence (IF) A549 cells were seeded on cover-glasses, treated (or not) with 1 U/ml GO for different exposure times and then processed for IF as previously described [18]. Primary Abs used in this study were: ap-Y416 c-Src (Cell Signaling, 1:200); ap-Y1173 EGFR (Cell Signaling, 1:50); aceramide*1(Alexis Biochem, 1:20). Secondary Abs were Alexa Fluor 488 or 555, either goat amouse or goat arabbit 1:200 dilution. Nuclei were stained with 49,6-diamidino-2- phenylindole (DAPI). Cholesterol in fixed cells was stained by 30 min. incubation with 50 mg/ml filipin (Sigma). Cells were mounted on slides using ‘‘fluoromount-G’’ (Southernbiotech) and images were acquired by confocal microscopy using an Olympus FluoView FV1000 or a LSM 5 Pascal Zeiss system with 4006 (air) or 6306 (oil) magnification objectives. Observations were repeated at least three times independently by different scientists. Acknowledgments We thank Dr. K. Omi, Fujirebio Inc., Tokyo (Japan), for providing the EGFR mAb 4-2. Author Contributions Conceived and designed the experiments: SF EMK TR TG. Performed the experiments: SF EMK ET CB MA TR. Analyzed the data: SF EMK TR TG. Contributed reagents/materials/analysis tools: SF EMK ET CB MA TR. Wrote the paper: SF EMK TG. 16. Kobayashi S, Boggon TJ, Dayaram T, Janne PA, Kocher O, et al. (2005) EGFR mutation and resistance of non-small-cell lung cancer to gefitinib. N Engl J Med 352: 786–792. 17. Jura N, Endres NF, Engel K, Deindl S, Das R, et al. (2009) Mechanism for activation of the EGF receptor catalytic domain by the juxtamembrane segment. Cell 137: 1293–1307. 18. Khan EM, Lanir R, Danielson AR, Goldkorn T (2008) EGF receptor exposed to cigarette smoke is aberrantly activated and undergoes perinuclear trafficking. FASEB J 22: 910–917. 19. Shen X, Kramer RH (2004) Adhesion-mediated squamous cell carcinoma survival through ligand-independent activation of epidermal growth factor receptor. Am J Pathol 165: 1315–1329. 20. Lambert S, Vind-Kezunovic D, Karvinen S, Gniadecki R (2006) Ligand-independent activation of the EGFR by lipid raft disruption. J Invest Dermatol 126: 954–962. 21. Bublil EM, Pines G, Patel G, Fruhwirth G, Ng T, et al. (2010) Kinase-mediated quasi-dimers of EGFR. FASEB J. 22. Chung I, Akita R, Vandlen R, Toomre D, Schlessinger J, et al. (2010) Spatial control of EGF receptor activation by reversible dimerization on living cells. Nature 464: 783–787. 23. Yu X, Sharma KD, Takahashi T, Iwamoto R, Mekada E (2002) Ligand- independent dimer formation of epidermal growth factor receptor (EGFR) is a step separable from ligand-induced EGFR signaling. Mol Biol Cell 13: 2547–2557. 24. Ravid T, Sweeney C, Gee P, Carraway KRK, Goldkorn T (2002) EGF receptor activation under oxidative stress fails to promote c-Cbl mediated down regulation. J Biol Chem 12: 12. 25. Ravid T, Heidinger J, Gee P, Khan E, Goldkorn T (2004) c-Cbl-mediated ubiquitinylation is required for EGF receptor exit from the early endosomes. J Biol Chem 279: 37153–37162. 26. Khan E, Heidinger J, Levy M, Lisanti M, Ravid T, et al. (2006) EGF receptor exposed to oxidative stress undergoes Src- and caveolin-1-dependent perinuclear trafficking. J Biol Chem 281: 14486–14493. 27. Reuter S, Gupta SC, Chaturvedi MM, Aggarwal BB (2010) Oxidative stress, inflammation, and cancer: How are they linked? Free Radic Biol Med. 28. Goldkorn T, Balaban N, Shannon M, Chea V, Matsukuma K, et al. (1998) H2O2 acts on cellular membranes to generate ceramide signaling and initiate apoptosis in tracheobronchial epithelial cells. J Cell Sci 111: 3209–3220. 29. Levy M, Castillo SS, Goldkorn T (2006) nSMase2 activation and trafficking are modulated by oxidative stress to induce apoptosis. Biochem Biophys Res Commun 344: 900–905. 30. Levy M, Khan E, Careaga M, Goldkorn T (2009) Neutral sphingomyelinase 2 is activated by cigarette smoke to augment ceramide-induced apoptosis in lung cell death. Am J Physiol Lung Cell Mol Physiol 297: 125–133. PLoS ONE | www.plosone.org 11 August 2011 | Volume 6 | Issue 8 | e23240