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-(-3morpholin-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)-6,7dimethoxyquinazolin-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-[(4methylpiperazin-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-2yl)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 l 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 M
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.