Hygromycin b ant hg 1

2 μg lentiviral vector containing sequences of interest, together with 0.75 μg pMD2.G (12259, Addgene), and 2.25 μg psPAX2 (12260, Addgene) were co-transfected into a 6-cm dish HEK-293 T cells using X-tremeGENE™ HP DNA Transfection Reagent (6366546001, Sigma-Aldrich). After 24 h, the medium may be replaced with complete DMEM medium. Virus-containing supernatant was collected 48 h and 72 h post transfection and centrifuged for 40 min at 3000 g at 4 °C. After that, viral supernatant was added to target cells in the presence of 8 μg/mL polybrene (H9268, Sigma-Aldrich). 48 h after infection, hygromycin B (ant-hg-1, Invivogen) selection at 1 mg/mL was added to obtain stable cell lines.

HEK293T and Flp-In T-REx 293 Cell Line (parental line; R78007; Thermo Fisher Scientific) were cultured in DMEM (high glucose, #12440-53; Gibco) supplemented with 10% FBS (#10500-64; Gibco), 2 mM L-glutamine (G7513; Sigma-Aldrich), 100 μg/ml Zeocin (ant-zn-1; InvivoGen), 15 μg/ml blasticidin (ant-bl-1; InvivoGen) at 37°C in a humidified 5% CO₂ atmosphere. The stable transfected Flp-In T-REx 293 cell lines (KRAS WT-APEX2, KRAS G12D-APEX2, KRAS G13D-APEX2, KRAS Q61H-APEX2, KRAS WT-GFP, KRAS G12D-GFP, KRAS G13D-GFP, KRAS Q61H-GFP, GFP only) were cultured in the same medium and selected by adding 100 μg/ml of hygromycin B (ant-hg-1; InvivoGen) to the media. For the induction of the stable transfected Flp-In T-REx 293 cell lines, 1 μg/ml tetracycline was added to the media.