Supplementary Figure 1



Supplementary Figure Legends

Supplementary Fig. 1 Induction and de-induction of transgene expression in C/L858R56 bitransgenic mice by quantitative RT-PCR, as measured for other lines in Figure 1. C, indicates a CCSP-rtTA mouse induced with doxycycline for 8 weeks.

Supplementary Fig. 2. The anti-EGFRL858R serum (see Materials and methods) specifically recognizes EGFRL858R, but not EGFRWT or EGFR(L747-S752. 293T cells were transiently transfected with expression vectors containing EGFRWT, EGFR(L747-S752, or EGFRL858R. Cells were harvested for immunoblot analyses using anti-EGFRL858R (top) and anti-total EGFR (bottom).

Supplementary Materials and Methods

Generation of Transgenic Mice

An initial version of the construct carried a 100bp segment of DNA, rich in restriction enzyme sites, between the TetO element and the ATG of EGFR. To prevent potentially deleterious effects of this element on the expression of the transgene, it was excised to create a second generation of EGFR transgenes. The transgenic lines described here were all derived from this second generation of EGFR transgenes. Tet-op-EGFR-mp1 transgenic constructs were excised from the parental vector using BssHII and then injected into fertilized (B6 X CBA) F2 eggs in the MSKCC Transgenic Core Facility. Transgenic founders were identified by PCR and Southern blotting of tail DNA. In the process of screening the founder lines for transgene expression, we noticed that not all C/L858R57 bitransgenic mice expressed the transgene. Southern blotting demonstrated that transgene expression was correlated with an array of transgenes in a head to tail configuration and not with other transgenic arrangements that segregated independently in this line (data not shown). For the studies described here, we used mice exclusively from line 57 that carry the doxycycline-inducible transgenic array. We sequenced DNA from two C/L858R and two C/DEL lungs with tumors and verified the presence of the expected mutation.

RT-PCR Analysis

Tissue samples were crushed and RNA was extracted using Trizol (Invitrogen) reagent. RNA was treated with DNase I (Invitrogen) to eliminate any contaminating DNA. RT-PCR reactions were carried out using the Superscript III One-Step RT-PCR with Platinum Taq system (Invitrogen). For control reactions, in the absence of RT, the Superscript RT/Platinum Taq mix was substituted with Platinum Taq (Invitrogen). Primers specific for human EGFR (that do not amplify mouse Egfr) used to amplify the transgene are “EGFR For” 5(-agcagtgactttctcagcaaca-3( and “EGFR Rev” 5(-ggctcggacgcacgagccgt-3(. Control reactions for actin RNA were carried out using the RT-PCR primer and control set (Invitrogen). Reverse transcription was carried out at 50°C for 30 min followed by 35 amplification cycles (94°C for 15 sec, 59°C for 30 sec, 72°C for 1 min) and a final extension (72°C for 10 min). For quantitative RT-PCR analysis, first strand cDNA synthesis (Invitrogen) was carried out on 1(g of DNase I (Invitrogen) -treated RNA in the presence and absence (control) of RT. The cDNA reactions were diluted 10-fold and 1 (l was used for each quantitative PCR reaction. Reactions were performed in a 10(l volume and contained 1(l of cDNA, 0.1(M primers and 5(l of Syber Green PCR Master Mix (Applied Biosystems) and were run in triplicate. Primers were as follows: “human EGFR For” 5(-cagcagagacccacactaccag-3(, “human EGFR Rev” 5(-gtcccgacagcttacacgaca-3(, “mouse Egfr For” 5(-acgtccgagaacacaaggac-3(, “mouse Egfr Rev” 5(-caagtacgggaaacgttaga-3(, “Ribosomal protein L37a For” 5(-tctgtggcaagaccaagatg-3(, “Ribosomal protein L37a Rev” 5(-gacagcagggcttctactgg-3(. Amplification conditions were as follows: 50°C for 2 min, 95°C for 10 min, 40 cycles of 95°C for 15 sec, 60°C for 1 min. Melting curve analysis was carried out to confirm that a specific amplification product was reproducibly obtained. Reactions were performed using the ABI7900 Sequence Detection System (Applied Biosystems). The relative expression levels of human or mouse EGFR between samples were determined by calculating the difference in the number of cycles (Ct) required to reach a threshold during the linear part of the PCR reaction for the test gene (human or mouse EGFR) compared to a control housekeeping gene (in our case, ribosomal protein L37a) for each sample. The following formula was used: ((Ct = [experimental sample Ct(EGFR) - experimental sample Ct(L37a)]-[reference sample Ct(EGFR) – reference sample Ct[L37a]). The relative expression level is calculated using the formula 2-((Ct (Pfaffl 2001).

Immunohistochemistry

The primary antibodies used for immunohistochemistry were anti–phospho (Ser10) histone H3 (used at a 1:200 dilution, #9701 Cell Signaling Technology); anti–cleaved (Asp175) caspase-3 (used at a 1:200 dilution, #9661 Cell Signaling Technology), anti-EGFR (K1492, Dako Pharmdx Kit), anti-EGFRL858R (used at a 1:300 dilution, see below for a description of this antibody), anti-prosurfactant protein C (used at a 1:400 dilution, AB3428 Chemicon), anti-clara cell protein (1:1000, AB3700 Chemicon), anti-phospho-EGF Receptor (Tyr992) (used at a 1:200 dilution, #2235 Cell Signaling Technology), anti-phospho- MAPK (used at a 1:100 dilution, #4376 Cell Signaling Technology), anti- MAP kinase antibody (used at a 1:100 dilution, #9102 Cell Signaling Technology), anti-phospho-Stat3 (used at a 1:100 dilution, #9135 Cell Signaling Technology), anti-Stat3 (used at a 1:100 dilution, #9132 Cell Signaling Technology), anti-phospho-Akt (used at a 1:100 dilution, #3787 Cell Signaling Technology) and anti-Akt (used at a 1:100 dilution, #9272 Cell Signaling Technology). Apoptotic and mitotic cells were identified by counting activated caspase 3 and phospho-histone H3 positive cells, respectively. At least 400 cells were scored from tumor-containing fields for each sample.

Antibodies and Immunoblotting

Tissue samples were crushed and lysed in lysis buffer (50 mM Tris·HCl pH 8.0, 150 mM sodium chloride, 5 mM magnesium chloride, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 40 mM sodium fluoride, 1 mM sodium orthovanadate) containing protease inhibitors (Roche Diagnostics). After quantitation by Bio-Rad protein assays, ~50 μg of each sample was separated by gel electrophoresis on 8% Tris-Glycine precast gels (Invitrogen) and transferred to nitrocellulose. Specific proteins were detected by using enhanced chemiluminescence (Amersham Pharmacia) and the following antibodies: anti-total EGFR (used at a 1:1000 dilution, BD Transduction Laboratories), anti-EGFRL858R (used at a 1:1000 dilution), anti-EGFR (clone LA22, used at a 1:1000 dilution, Upstate), anti-actin (used at a 1:5000 dilution, Sigma), HRP-conjugated anti-rabbit Ig (used at a 1:5000 dilution, Amersham Pharmacia), and HRP-conjugated anti-mouse IgG (used at a 1:2000 dilution, Roche Applied Science).

MR Imaging

Initial Fast Spin Echo scout images in 3 orthogonal directions were acquired for positioning the animal. Subsequently axial and coronal multi-slice lung T1 Spoiled Recalled Gradient Echo images were acquired with a slice thickness of 0.7 mm and a slice gap of 0.2 mm. The TR (time of repetition) was approximately 247 msec and the TE (echo time) was 2.5 msec. An acquisition matrix of 256 x 192 was used, with a field of view of 3 x 2.5 cm or 3 x 2.2 cm for coronal and axial images respectively, yielding a corresponding spatial resolution of 117 x130 (m and 117 by 115 (m. Each image used 8 excitations per phase-encoding step with a total scanning time of 6.4 minutes.

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