Amg510

All cells were cultured at 37°; air; 95%; CO₂, 5%. H358 (NCI-H358), H23 (NCI-H23), H1792 (NCI-H1792) and 293T cells were obtained from American Type Tissue Culture (ATCC). Cells were regularly checked for mycoplasma contamination by polymerase chain reaction^{64 (link)} and found to be negative. H358 sgRB1#4 parental and resistant cell lines were verified by STR profiling (Labcorp, Burlington, NC, USA). LUAD cells were grown in RPMI−1640 medium (Gibco, 11875119) supplemented with 10% fetal bovine serum (FBS) (Gibco, 12483020) and 1% Pen/Strep (Gibco, 15140-122). 293T cells were grown in DMEM medium complete with 10% FBS and 1% Pen/Strep (Gibco, 15140-122). For cells and experiments with doxycycline-inducible constructs, cells were grown in RPMI-1640 medium (Gibco, 11875119) supplemented with 10% tetracycline-free FBS (Clontech, 631101) and Pen/Strep (Gibco, 15140-122). Doxycycline hyclate (Sigma-Aldrich, D9891) was added to cells at 200 ng/mL when indicated. Trametinib (Selleckchem, S2673), SCH772984 (Selleckchem, S7101), AMG 510 (Selleckchem, S8830), SB 747651 A (Tocris, 4630), MK-2206 (Selleckchem, S1078), NSC 23766 (Selleckchem, S8031), dabrafenib (Selleckchem, S8031), infigratinib (Selleckchem, S2183) and N-acetyl-L-cysteine (Sigma-Aldrich, A7250) were added to cells when indicated. Experiments were performed on cells between passages 4-20.

Single-cell expression profiling of cells perturbed by a KRAS(G12C) inhibitor was performed as follows: NCI-H3122, NCI-H358 and SW480, with KRAS genotypes of wild type, G12C, and G12V, respectively, were trypsinized after 24 h of culture. Then, cells were washed with PBS, and plated in poly-d-lysine-coated wells of a 12-well plate at a density of 1000 cells per well in 1 ml of medium. Four wells per cell line were seeded for different drug concentrations, the cells were evenly distributed by rocking the plate and the plate was incubated at 37°C for 8 h to allow attachment. After incubation, 700 μl of the medium was removed and another 700 μl of medium containing various concentrations of AMG-510 (Selleckchem, Cat. No. S8830) was added to the wells while minimizing any disturbance of the cells adhering to the bottom. After 6 h of drug treatment at 37°C, 700 μl of the medium was removed and 700 μl of the incubation buffer containing 20 000 beads was added to the well. The beads were spread evenly by rocking the plate and incubated for 15 min to allow them to settle. Cells were lysed by the diffusive lysis method and the preparation of single-cell libraries was performed as described above.

MiaPaCa-2, NCI-H2122, NCI-H358, and Panc-1 cells were purchased from ATCC in 2012, 2013, 2021, and 2012, respectively. NCI “Rasless” mouse embryonic fibroblast (MEF) cell lines (KRAS 4B WT, G12C, G12D, G12V) were obtained from the NCI (Rockville, MD) in 2019. MiaPaCa-2, Panc-1, and all the MEF cell lines were maintained in DMEM (Thermo Fisher Scientific), while NCI-H2122 and NCI-H358 were maintained in RPMI1640 (Thermo Fisher Scientific), supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin in a 5% CO₂ atmosphere at 37°C. The cell lines have been tested and authenticated in a core facility of the Applied Genomics Technology Center at Wayne State University. The method used for testing was short tandem repeat (STR) profiling using the PowerPlex 16 System (Promega). Mycoplasma testing was routinely performed on the cell lines using PCR. All experiments were performed within 20 passages of the cell lines. MRTX1257 (ChemieTek), AMG510, MRTX849 (Selleck Chemicals LLC), and selinexor (Karyopharm Therapeutics) were dissolved in DMSO to make 10 mmol/L stock solutions. The drug control used for in vitro inhibitor experiments was cell culture media containing 0.1% DMSO.