SUPPLEMENTARY MATERIALS AND METHODS

Cell Culture and Transfection

For stimulation studies with EGF (100 ng/ml) (Calbiochem, Darmstadt, Germany), cells were cultured in serum-free medium containing 0.5% BSA (Invitrogen, Carlsbad, CA) for up to 16 hours. For transfection studies, COS-7 cells were transfected using FuGene™-6 reagent (Roche, Indianapolis, IN) according to the manufacturer's instructions. Transient transfection using COS-7 cells with EGFR plasmid constructs was first optimized using different ratios of transfection reagent to plasmid DNA. The optimal transfection condition with 3:2 ratio of FuGene™-6 transfection reagent (μl) to DNA (μg) was used for establishing stable transfectant cell lines using G418 (500 µg/ml) selection. Parental COS-7 cells had negligible endogenous EGFR expression (data not shown).

Cell Proliferation and Cytotoxicity Assays

Cell proliferation and cytotoxicity assays were performed using tetrazolium compound based CellTiter 96® AQueous One Solution Cell Proliferation (MTS) assay (Promega). To test for cellular cytotoxicity induced by EGFR TKIs, mutant EGFR stably transfected COS-7 cells (L858R and L858R+E884K) were cultured in 5% FBS media with EGF stimulation (20 ng/ml). MTS assay was then performed according to the manufacturer’s instruction at 72 hours after treatment with indicated EGFR TKI (gefitinib or erlotinib) at indicated concentrations. For cell proliferation assay, stably transfected COS-7 cells were cultured for 5 days under regular growth conditions (10% FBS), with MTS cell viability assay performed daily according to the manufacturer’s instructions.

EGFR TKIs and MET TKIs

The EGFR reversible TKIs gefitinib (N-(3-cholro-4-fluoro-phenyl)-7-methoxy-6-(-3-morpholin-4-ylpropoxy)quinazolin-4-amine)(Iressa®), and erlotinib (N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine)(Tarceva®), as well as the dual EGFR/ERBB2 reversible TKI lapatinib, N-[3-Chloro-4-[(3-fluorophenyl)methoxy]phenyl]-6-[5-[(2-methylsulfonylethylamino)methyl]-2-furyl]quinazolin-4-amine (Tykerb®), were obtained commercially, dissolved in dimethylsulphoxide (DMSO) and diluted to the indicated concentrations as previously described (Choong et al., 2006). The following tyrosine kinase inhibitors were purchased from Calbiochem, Inc. (Darmstadt, Germany): (a) 4557W, 4-(4-benzyloxyanilino)-6,7-dimethoxyquinazoline, a dual EGFR/ERBB2-TKI (reversible); (b) GW583340, N-(3-chloro-4-[{3-fluorophenyl}methoxy]phenyl)-6-(2-[{2-[methyl-sulfonyl]ethyl}amino]methyl)-4-thiazolyl)-4-quinazolinamine dihydrochloride, a dual EGFR/ERBB2-TKI (reversible); (c) Tyrphostin AG1478, N-(3-chlorophenyl)-6,7-dimethoxyquinazolin-4-amine hydrochloride, an EGFR-TKI (reversible) (d) CL-387,785, N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]2-butynamide, an irreversible anilinoquinazoline EGFR-TKI.

The small molecule selective reversible MET-TKIs used in this study include: (a) SU11274, [(3Z)-5-(2,3-dihydro-1H-indol-1-ylsulfonyl)-3-({3,5-dimethyl-4-[(4-methylpiperazin-1-yl)carbonyl]-1H-pyrrol-2-yl}methylene)-1,3-dihydro-2H-indol-2-one] (purchased from Calbiochem), and (b) PHA665752, (3Z)-5-[(2,6-dichlorobenzyl)sulfonyl]-3-[(3,5-dimethyl-4-{[(2R)-2-(pyrrolidin-1-ylmethyl)pyrrolidin-1-yl]carbonyl}-1H-pyrrol-2-yl)methylene]-1,3-dihydro-2H-indol-2-one (kind gift from Pfizer, Inc; La Jolla, CA). For tyrosine kinase inhibition study, cells were incubated with the above small molecule inhibitors at the indicated final concentrations for four hours, followed by EGF stimulation (100 ng/ml, 10 minutes).

SSCP and DNA Sequencing

PCR: PCR was performed in a 25 ml reaction mix containing 15 mM Tris-HCl, pH 8.0, 50 mM KCl, 2.5 mM MgCl2, 0.2 mM dNTP, 0.5 units AmpliTaq Gold, 5 % DMSO, 0.1 mM forward and reverse primers. The cycling condition consist of an initial denaturation at 950C for 10 min, and 40 cycles of 950C for 15 seconds, 550C for 30 seconds and 720C for 30 seconds, and followed by 5 min at 720C. SSCP: It was performed as described by Xie et al. (Xie et al., 1997).

Direct DNA sequencing was performed at the Sequencing Core Facility at the Department of Genetics, Case Western Reserve University. The sequences of the primers are available upon request.