H1650

For all experiments we used the following human cancer cell lines: (U87 (ATCC- HTB-14™), U251 (ATCC)), neuroblastoma (SH-SY5Y (ATCC-CRL-2266™)), lung adenocarcinoma (A549 (ATCC- CCL-185™), H460 (ATCC- HTB-177™), H1650 (ATCC-CRL-5883™), H1975 (ATCC-CRL-5908™)), breast cancer (MCF-7 (ATCC-HTB-22™), MDA-MB-231 (ATCC-HTB-26™)), human astrocyte (Sciencell-1800), human HBE1(Gift from Dr. Amir Zeki lab, UC Davis), and human MCFD10A (Gift from Dr. Amir Zeki lab, UC Davis). Cancer cells were cultured in DMEM (high glucose) supplemented with FBS (10%), penicillin (1%), and streptomycin (1%). Human astrocytes were cultured in DMEM supplemented with FBS (10%), penicillin (1%), and streptomycin (1%). HBE1 cells were grown to 90% confluency on a 100 mm cell culture plate in serum-free medium containing Ham’s F12/DMEM (1:1), 15 mM NaHCO₃, 15 mM Hepes (pH 7.4), with these factors: transferrin (5 μg/mL), insulin (5 μg/mL), cholera toxin (10 ng/mL), epidermal growth factor (10 ng/mL), dexamethasone (0.1 μM), bovine hypothalamus extract (15 μg/mL). Cells were then transferred to 6-well plates in submerged media conditions. MCF10A were grown in Lonza CC-3150 MEGM per ATCC’s recommendation without gentamicin/amphotericinplus 100 ng/mL cholera toxin. Cells were then transferred to 6-well plates.

All cell lines (A549, H441, H460, H1650, Calu 1, and Calu 6) were obtained from the ATCC (Manassas, VA, USA). A549 and H441 cells represented preinvasive stages, while H460, H1650, Calu 1, and Calu 6 cells represented advanced types [37 (link)–40 (link)]. Cells were grown in RPMI1640 media supplemented with 10% (v/v) fetal bovine serum (FBS), penicillin, and streptomycin, in a humidified atmosphere of 5% CO₂ and 95% air at 37° C. The cells were tested and authenticated by short tandem repeat analysis. Stably transformed cells were selected using geneticin (1000 μg/mL) and collected by cell sorting three times (>95% purified) with a fluorescent protein marker (ZsGreen1) to improve cell purity [41 ].

All animal work was conducted in accordance with a protocol approved by the Institutional Animal Care and Use Committee at Weill Cornell Medical College. Wild type C57BL/6J and p53^f/f (B6.129P2-Trp53^tm1Brn/J) mice were obtained from The Jackson Laboratory (Bar Harbor, Maine), and K-ras (B6.129S4-Kras^tm4Tyj/J (LSL Kras-G12D)) mice from The National Cancer Institute (Bethesda, Maryland). The p53^f/f and K-ras mice were then bred together for 3 generations to get Kras-Het; p53f/f mice.
The HKP-1M cell line was established by culturing cells derived from lung adenocarcinomas from mice which constitutively express oncogenic K-ras and have loss of function of p53. Cells were cultured in DMEM with 10% fetal bovine serum. The human non-small cell lung cancer cell line (H1650) was obtained from ATCC and cultured in RPMI with 10% fetal bovine serum.

Human lung cancer cells lines (A549, H460, H1650, H522, H520 and H1299) were purchased from ATCC. They were cultivated in RPMI medium supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mM l-glutamine. Cells were grown at 37 °C in 5% CO₂ atmosphere.

Two NSCLC cell lines H1650 (ATCC CRL-5883) and H1975 (ATCC CRL-5908), and human embryonic kidney 293 cell line (HEK293, ATCC CRL-1573), were purchased from American Type Culture Collection (Manassas, VA). The HEK293 cell line has wild-type EGFR and both H1650 and H1975 cell lines have EGFR mutations (heterozygous exon 19 delE746-A750 for H1650, heterozygous exon 21 L858R, and exon 20 T790M for H1975^{[16 (link)]}). Cells were grown in Roswell Park Memorial Institute growth media supplemented with 10% fetal calf serum, 30 ng/mL EGF, 10 U/mL penicillin, and 10 μg/mL streptomycin at 37°C and 5% CO₂.
DNA was extracted from formalin-fixed paraffin embedded tumors using the QIAamp DNA FFPE Tissue Kit and from cell lines using the QIAamp DNA Mini Kit (Qiagen).

Immortalized bronchial epithelial cells (BEAS-2B) and nine human lung cancer cell lines (A549, H1299, H1650, H1975, H2030, H292, H460, H838, SK-LU-1) were purchased from ATCC (American type culture collection). All cell lines were confirmed to be free of mycoplasma contamination. BEAS-2B and H292 cells were cultured in Dulbecco's Modified Eagle Medium with 10% fetal bovine serum (FBS). A549, H1299, H1650, H1975, H2030, H460, H838 and SK-LU-1 cells were cultured in RPMI 1640 medium with 10% FBS. All cell lines were maintained at 37°С in a 5% CO₂ incubator. Cell lines were immediately expanded upon receipt and aliquots frozen to allow the cell lines to be restarted every three to four months from the same batch of cells. Cell phenotypes were verified in every experiment.