Culturing and Characterizing Engineered HeLa and Flp-In 293 T-REx Cell Lines

HeLa cells were cultured in MEM (catalogue no. ECB2071L, Euroclone) supplemented with 10% inactivated fetal bovine serum (FBS) (catalogue no. ECS0180L, Euroclone), 2 mM glutamine (catalogue no. ECB3000D, Euroclone), penicillin (100 IU ml⁻¹) and streptomycin (100 μg ml⁻¹) (catalogue no. ECB3001D, Euroclone) and maintained at 37 °C and 5% CO₂. RagC KO HeLa cells and RagA KO HeLa cells were previously generated and described in ref. ^{7 (link)}. Flp-In 293 T-REx cells (catalogue no. R78007 Thermo Fisher) were grown in DMEM (catalogue no. D6429 Sigma-Aldrich), supplemented with 10% (vol/vol) FBS (catalogue no. 10270 Thermo Fisher), 100 U ml⁻¹ penicillin and 100 µg ml⁻¹ streptomycin (catalogue no. P0781 Sigma-Aldrich), 100 µg ml⁻¹ Zeocin (catalogue no. ant-zn-5b InvivoGen, Toulouse, France) and 15 µg ml⁻¹ Blasticidin (ant-bl-5b InvivoGen). Cell lines were validated by morphological analysis and routinely tested for absence of mycoplasma.