Dmem d glucose medium

Human breast carcinoma cell lines MDA-MB-231, BT549, MDA-MB-435, MDA-MB-468, SKBR3, BT474, T47D, MCF7, and ZR75 were obtained from the American Type Culture Collection–LGC Standards Ltd. Partnership. All cell lines were cultured in DMEM d-glucose medium (Gibco) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin, except BT459 cells, which were cultured in supplemented RPMI medium (Gibco). All cells were cultured at 37°C and in a 5% CO₂ humidified atmosphere. For lentiviral infection, human embryonic kidney–293 T cells were transfected with pLKO lentiviral vectors and plasmids encoding lentiviral particles using standard methods. pLKO sh_CPEB2 plasmids were obtained from Sigma-Aldrich MISSION shRNA library (clones TRCN0000149728 and TRCN0000149778). Recipient cells were transduced with the viral medium and selected with puromycin (2 μg ml⁻¹) for 72 hours.

The B16F10 murine melanoma cell line was obtained from ATCC. B16F10 cells expressing OVA‐GFP were provided by D. Sancho and have been described previously (Sancho et al, 2008 (link)). B16F10 TGL cells were generated by infecting B16F10 cells with retrovirus containing the TGL (Thymidine kinase‐GFP‐Luciferase, provided by R. Gomis) plasmid. Briefly, HEK–293T cells were transfected with TGL vector and plasmids encoding retroviral particles using standard methods. B16F10 cells were subjected to two consecutive rounds of infection and expanded for 7 days without selection. At day 7, GFP⁺ cells were isolated by fluorescence‐activated cell sorter (FACS) and expanded. Cells were cultured in DMEM d‐glucose medium (Gibco) supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and 2 mM L‐glutamine (Gibco). Mouse colorectal tumor organoids (MTOs) carrying patient‐specific oncogenic mutations and expressing luciferase have been previously described (Tauriello et al, 2018 (link)).