Imdm medium

Bovine serum albumin (BSA), NADPH, human glutathione reductase (hGR), oxidized glutathione (GSSG) and 5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB), IMDM medium (Iscove’s Modified Dulbecco’s Medium), sodium bicarbonate, hypoxanthine, thymidine, bathocuprione sulfonic acid, cysteine, β-mercaptoethanol, heat-inactivated Calf serum, triton-X 100, anti-His tag antibody and protease inhibitors cocktail were purchased from Sigma-Aldrich (St. Louis, USA); oxidized trypanothione (TS₂) was purchased from Bachem (Bubendorf, Switzerland); NADPH-Glo kit and CellTiter-Glo were purchased from Promega (Madison, WI).

The murine αTC1 cell line was obtained from ATCC (Cat#CRL-2934, RRID:CVCL_B036). Cells were grown in low-glucose DMEM medium (Biowest L0066) supplemented with 10% FBS, 50 U/mL penicillin and 50 μg/mL streptomycin. HAP1 cells (Horizon discovery) were grown in IMDM medium (Sigma I6529) supplemented with 10% FBS, 50 U/mL penicillin and 50 μg/mL streptomycin. The Lenti-X™ 293 T cell line was purchased from Takara Bio (632180). Cells were grown in high-glucose DMEM medium (Sigma D5796) supplemented with 10% FBS, 1 mM sodium pyruvate, 50 U/mL penicillin and 50 μg/mL streptomycin.

MV4-11 cells were cultured in IMDM medium (Sigma-Aldrich) supplemented with 20% FCS, 1% L-glutamine, and 1% penicillin/streptomycin. Cells were constantly maintained at 37°C in 5% CO₂. The Flt3-ITD phosphokinase inhibitor was added to cell cultures of MV4-11 cells in 2 different concentrations for 15 minutes before the cells were harvested for mRNA extraction (0.1 mM and 1 mM) (LC Laboratories, Woburn, MA, USA).

HMC-1.1 were used for experiments on mast cells [83 (link)]. An aliquot of HMC-1.1 cells had previously been generously gifted by Dr Butterfield to PTKS allowing us to conduct in vitro mast cell investigations. Cells were cultured in IMDM medium (Gibco, Paisley, UK) supplemented with 10% calf serum (Hyclone, Logan, UT, USA), using 162 cm³ cell culture flasks (Corning Incorporated, Costar^®, Corning, NY, USA) and incubated at 37 °C with 5% of CO₂. Cells were split and collected in 6 wells culture plates (Corning Incorporated, Costar^®, Corning, NY, USA) at a density of 1 × 10⁶ cells/mL. Each well contained 3 mL of cell suspension. The HMC-1.1 cells were maintained in IMDM medium, supplemented with 10% calf serum, plus α thioglycerol (Sigma, Gillingham, UK). Cell counting was performed using Countess II Life Technologies Cell Counter (Countess™ Invitrogen, Waltham, MA, USA), by Trypan blue staining.

For generation of lenti‐ or retroviral particles, 1.25 × 10⁵ HEK 293T cells were seeded per well in 24‐well plates. For transfection the plasmid of interest (400 ng), the viral packaging plasmid pSPAX2 (200 ng) for lentiviruses or HIT‐60 for retroviruses, and pVSVg (100 ng) for pseudotyping were used in 20 μL IMDM medium (Sigma‐Aldrich), mixed with 20 μL IMDM medium containing 1.2 μL polyethylenimine (PEI), incubated for 30 min at room temperature and added drop‐wise to the 293T cells. Virus particles were harvested after 40–48 h post transfection. Viral supernatants were mixed with polybrene (16 μg·mL⁻¹ final concentration). Target cells were transduced using spin infections in 1.5 mL reaction tubes at 37 °C and 300 g for 90 min. After puromycin selection (2 μg·mL⁻¹) for 2 days or cell sorting (via dsRed marker), 500 cells were seeded in 5 mL Baf3 medium containing 2% Methylcellulose and plated in a 10 cm bacterial plate. Single‐cell colonies were picked after 9–10 days.

Human uveal melanoma cell lines 92.1 (BAP1-pos, GNAQ-mut),⁵⁰ (link) OMM1 (BAP1-pos, GNA11-mut),⁵¹ (link) Mel270 (BAP1-pos, GNAQ-mut),⁵² (link) XMP46 (BAP1-neg, GNAQ-mut),⁵³ (link) MM28 (BAP1-neg, GNA11-mut),⁵³ (link) human conjunctival melanoma cell lines CRMM1 (BRAF-mut),⁵⁴ (link) CRMM2 (NRAS-mut),⁵⁴ (link) and human melanoma cell line OCM3 (BAP1-pos, BRAF-mut)⁵⁵ (link) were studied. An overview of studied cell lines and their genetic mutations is provided in Supplementary Table S1.⁵⁶ (link)
Cell lines 92.1, OCM3, OMM1, and Mel270 were grown in RPMI 1640 medium (Gibco, Life Technologies Co.) supplemented with 10% fetal bovine serum (FBS; Gibco, Life Technologies Co.) and 1% antibiotics (10,000 units/mL Penicillin, 10,000 ug/mL Streptomycin; Gibco, Life Technologies Co.). Cell lines XMP46 and MM28 were grown in IMDM medium (Sigma-Aldrich, UK) supplemented with 20% FBS and 2% antibiotics. Cell lines CRMM1 and CRMM2 were grown in F-12K medium (Gibco, Life Technologies Co.) supplemented with 10% FBS and 1% antibiotics. Cells were incubated in a humidified atmosphere of 5% CO2 at 37°C. Cells were protected from light using aluminum foil, and the experiments were performed under dimmed lights.