Wz4002

Cell survival was assayed by clonogenic assays, cell proliferation assays, or monolayer growth. For clonogenic assays, after the H1975 cells were exposed to normoxia or hypoxia for 4 weeks, cells were seeded at 500/well in 6-well plates and treated with AZD9291 or WZ4002 (Selleckchem) at the indicated concentrations. Colonies were washed with 0.9% saline solution and stained with crystal violet 10–14 days later. Colonies consisting of >50 cells were counted. Colony formation was normalized to plating efficiency of the untreated cells. Errors bars in the survival analysis are based on three independent experiments. For cell proliferation assay, after the H1975 cells were exposed to normoxia or hypoxia for 4 weeks, 1X10⁵ cells were seeded in 100 mm dish and treated with AZD9291 or WZ4002 (Selleckchem) at 1 μM. Cell growth inhibition by AZD9291 and WZ4002 in H1975 N and H1975 H cells were measured by serial cell counts after 3, 6 and 9 days treatment. To quantify survival by monolayer growth, cells were seeded at a defined density in 6-well plates and treated with AZD9291 at the indicated concentrations for 5 days. Cells were stained with crystal violet for monolayer visualization and then the stain was dissolved in 70% ethanol and quantified by plate reader.

The JAK1/2 inhibitor AZD1480 was provided by AstraZeneca. The type II JAK2 inhibitor BBT594 was provided by Novartis. INCB18424 and EGFR TKI erlotinib were purchased from Chemietek. Pan-JAKi P6 was purchased from Calbiochem. The EGFR T790M–specific inhibitor WZ4002 was purchased from Selleck Chemicals. For in vitro experiments, the inhibitors were dissolved in 100% dimethyl sulfoxide (DMSO) to prepare a 10 mM stock and stored at −20°C. For in vivo experiments, the indicated inhibitors were formulated daily in purified, sterile water supplemented with 0.5% methyl cellulose and 0.1% Tween 80.

The human NSCLC cell lines (HCC827 and H1975) and NIH 3T3 (mouse embryonic fibroblast cell line) were obtained from the American Type Culture Collection (Rockville, MD). PC-9 cells were a gift from F. Koizumi and K. Nishio (National Cancer Center Hospital, Tokyo, Japan). PC-9/GR (gefitinib-resistant cell line) and PC-9/ER (erlotinib-resistant cell line) cells were established as part of a previous study (33 (link)). Cells were cultured in RPMI 1640 medium containing 10% FBS, 2 mmol/L L-glutamine, and 100 units/mL of penicillin and streptomycin, and maintained at 37 °C in a humidified chamber containing 5% CO₂. Osimertinib, CO-1686 and WZ4002 were purchased from Selleck Chemicals (Houston, TX). The cell lines used were authenticated by STR analysis and confirmed to be mycoplasma free using standard methods.