Imdm culture medium

PACO cells were grown to 80% confluency. The transfection reagent Dharmafect 4 (Thermo Scientific), nOn-Targeting (NT-control) and CYP3A5, HNF4A or NR1I2 siRNA (On-Target plus SMARTpool Thermo Scientific, Supplementary Table 12) were pre-incubated at RT for 5 min at a ratio of 1:4 in IMDM culture medium (Gibco). For the HNF4A and NR1I2 double knockdown, the individual siRNAs were pre-incubated together at a ratio of 1:8 in IMDM culture medium (Gibco). Dharmafect 4 was then combined with the siRNAs and incubated for further 20 min at RT. The mixture was then added to the PACO culture medium. The culture medium was aspirated and the transfection agent-RNA complex mixture was added to the monolayer. Flasks were incubated at 37 °C for 72 h until further analysis.

NK cells were purified from TILN and TFLN cell suspensions in selected experiments, using the NK cell Isolation kit negative selection and VarioMACS (Miltenyi Biotec) according to the manufacturer’s instructions for the depletion of non-NK cells. Briefly, after thawing, TILN and TFLN cell suspensions in RPMI 1640 medium were cultured overnight at 37°C in a 5% CO₂ humidified atmosphere. After a preliminary passage through a Ficoll column (Biopaque) to remove debris and dead cells, the purity of NK cell preparations, determined by cytofluorometric analysis, was above 95%. Freshly enriched NK cells were suspended in IMDM culture medium (Life Technology) supplemented with Penicillin (100 IU/ml) and Streptomycin (100 μg/ml), 10% FBS (Invitrogen). Peripheral blood lymphocytes (PBL) were isolated from blood of patients with melanoma or of healthy donors by density gradient centrifugation over Ficoll-Paque (Biopaque).