Hmgs 2

QualiCell^® human melanoblast stem cells (Creative Bioarray, New York, NY, USA) were maintained in culture in Medium 254 (M254, Invitrogen, Waltham, MA, USA) with propidium monoazide-free human melanocyte growth supplement (HMGS-2, Invitrogen) in an incubator with 5% humidified atmosphere at 37 °C. Melanoblasts from passages 3 to 7 were used for experiments. The medium was changed every 2–3 days. For subculture, cells were washed with phosphate buffered saline (PBS), detached with Accutase (Innovative Cell Tech., San Diego, CA, USA), and passaged at a plate 1:3 plate ratio when the cells reached 80–90% confluence.
Melanoblasts were cultured in melanocyte differentiation medium, to produce melanocytes. The differentiation medium consisted of M254, HMGS-2, 10 nM α-MSH (Sigma Aldrich, St. Louise, MO, USA), 10 nM 12-O-tetradecanoyl-phorbol-13-acetate (TPA; Sigma Aldrich), and 20 μM forskolin (Sigma Aldrich). Experiments progressed once the melanoblasts had been treated with the differentiation medium for 3 days when the medium was replaced with M254 medium, and the cells were exposed to ELF-EMFs.

Rabbit melanocytes were isolated from the dorsal skin tissue of Rex rabbits as described by Chen et al. (2019) (link). The cells were cultured in M254 medium (Gibco, Carlsbad, CA, USA) supplemented with 1% human melanocyte growth supplement-2 (HMGS-2, Gibco, Carlsbad, CA, USA) and maintained in an incubator at 37 °C in an atmosphere containing 5% CO₂. The seeded cells were cultured in 6-well plates to 70–90% confluence, then overexpression of KIT achieved using Lipofectamine™ 3000 Reagent (Invitrogen, Carlsbad, CA, USA), in accordance with the manufacturer’s instructions. A 2 µg quantity of KIT DNA plasmid in Opti-MEM™ medium (Gibco, Carlsbad, CA, USA) was added to 6 µL Lipofectamine™ 3000 diluted with Opti-MEM™ then incubated for 10–15 mins at room temperature. The DNA-lipid complex was added cells and incubated for 48 h with M254 medium, prior to qRT-PCR analysis.

Primary rabbit skin melanocytes were separated by two-step enzymatic digestion from 1.5 × 1.5-cm² sections of back skin from 2-month-old rabbits. White hair and subcutaneous fat tissue were removed and the tissues were digested in 0.25% Dispase II enzyme solution at 4 °C for 14–16 h. The epidermis and dermis were peeled off with fine sputum and digested in 0.25% trypsin at 37 °C for 8 min. Digested cells were cultured in M254-specific medium containing 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA). The medium was changed every 2 days to observe the growth state of the melanocytes. When cell growth reached ~90%, cells were digested with 1 mL of 0.25% trypsin for 1 min. Subsequently, cells were passaged at a ratio of 1:2 in 1% HMGS-2 (Human Melanocyte Growth Supplement-2, PMA-Free) (Gibco, Carlsbad, CA, USA) of M254 complete medium. Cells were maintained at 37 °C in a 5% CO₂ incubator.

Metastatic melanoma (MM) cell lines were established as described in Flørenes et al.,¹⁸ and WM melanoma cell lines from the Wistar cell line collection (Philadelphia, PA, USA) were a kind gift of Meenhard Herlyn (cells can be obtained from Rockland Immunochemicals, Inc., PA, USA; catalog numbers are provided in Table S1). Melanoma cells were cultured in RPMI 1640 medium (Bio Whittaker, Verviers, Belgium) supplemented with 5% fetal bovine serum (cat.no F7524, Sigma), 2‐mM l‐glutamine (cat. No. 17‐605E, Lonza). Primary human melanocytes (NHM9, NHM134, and NHM160) were obtained as previously described ¹⁹ and cultured in 254CF melanocyte media (cat. No. M254CF500) purchased from Gibco Life Technologies (California, USA), supplemented with calcium chloride, HMGS‐2 (human melanocytes growth supplement‐2) (cat. No. S0165, Gibco), and 10‐ng/ml PMA. All cells were maintained at 37°C in a humidified 5% CO₂ atmosphere.
Melanoma lymph node metastases were obtained from patients operated at the Norwegian Radium Hospital, Oslo University Hospital. The study design was approved by Norway Regional Committee for Medical and Health Research Ethics (approval number 2015/2434). Written informed consent was obtained from all patients included in the study.

Primary human epidermal melanocytes (NHEMs) were isolated from human foreskin specimens obtained during circumcision surgery and cultured in Medium 254 (M254500) supplemented with HMGS-2 (S0163) and 1% antibiotic-antimycotic (Gibco, Grand Island, NY, USA). This study was approved by the ethics committee of Shanghai General Hospital (2017KY005) and conducted according to the principles of the Declaration of Helsinki. Cells were cultured in a humidified atmosphere with 5% CO₂ at 37°C. Second- and fourth-passage NHEMs were used in all experiments. AS-IV (1044108), 6-formylindolo [3,2-b]carbazole (FICZ, SML1489), 8-MOP (M3501), AKT inhibitor VI (124013), FH535 (F5682), and CH223191 (C8124) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and were dissolved in DMSO.