Nu7026

All cell lines were routinely tested for Mycoplasma and profiled and identified by RNA-seq and the GenePrint 10 system (Promega) at periodic intervals during the course of this study. All M series cell lines were established from patient-derived tumors at the UCLA with institutional review board approval. All M cell lines with acquired MAPKi resistance were derived in the Lo Laboratory and published previously (3, 5, 8, 9, 11, 22, 40, 67 (link)). We maintained H358 (ATCC) in RPMI 1640 (Gibco) with 10% heat-inactivated FBS (Omega Scientific, FB-02) and 2 mmol/L glutamine in a humidified, 5% CO₂ incubator at 37°C; all other cell lines were maintained in high-glucose DMEM (Omega Scientific, DM-22) with 10% heat-inactivated FBS (Omega Scientific, FB-02) and 2 mmol/L glutamine in a humidified, 5% CO₂ incubator at 37°C. We obtained inhibitors from the following sources: PLX4032 (Plexxikon), AZD6244 (Selleck Chemicals), trametinib (LC Laboratories), NU7026 (Abcam, ab120970), ABT888 (Enzo, ALX-270-444-M005), VX984 (MCE, HY-19939S), AZD7648 (TargetMol, T7122), olaparib (LC Laboratories, 763113-22–0), MRTX849 (Selleckchem, S8884), AMG510 (Selleckchem, S8830), and BGB-283 (BeiGene, via a Material Transfer Agreement with UCLA). All inhibitors were dissolved in DMSO and stored at −20°C.

MRC-5 and A549 cells were plated into 96-well dishes at a density of 5×10⁴ cells/well. NU7026 and CGK733 (Abcam, Cambridge, UK) were added to each well at a final concentration of 5–50 µM, and incubated for 48 h. Thereafter, MTT (final concentration, 5 mg/ml) was added to each well. The medium was then removed, and the formazan crystals were dissolved by adding 150 µl dimethyl sulfoxide. The absorbance at 490 nm was subsequently measured in a microplate reader (Infinite M200; Tecan Group Ltd., Männedorf, Switzerland) (16 (link),17 (link)).