Leibovitz s l 15

Human melanoma cell lines Sbcl-2, WM3211, WM1366, WM793, WM1158, and WM9 (a generous gift from Dr. M. Herlyn, Wistar Institute, Philadelphia, USA) derived from RGP (Sbcl-2), VGP (WM3211, WM1366, WM793), and melanoma MET (WM1158, WM9) were maintained in a culture medium consisting of MCDB153 (Sigma-Aldrich, Steinheim, Germany) with 20% Leibovitz’s L-15 (PAA Laboratories, Coelbe, Germany), 2% FCS, 1.68 mM CaCl₂ (Sigma), and 5 µg/ml insulin (Sigma-Aldrich, Steinheim, Germany) at 37 °C and 5% CO₂. NHEM were derived from neonatal foreskin (NHEMs, PromoCell, Heidelberg, Germany) and were cultured in melanocyte growth media M2 at 37 °C and 5% CO₂.

The human melanoma cell lines 501mel, HMB2 and Mel Im were derived from metastases of malignant melanomas. Cells were maintained in DMEM supplemented with penicillin (400 U/ml), streptomycin (50 μg/ml), L-glutamine (300 μg/ml) and 10 % fetal calf serum (FCS) and split at a 1:5 ratio every three days. The cells were cultured under a humidified atmosphere of 8 % CO₂ at 37°C. Further human melanoma cell lines were a kind gift by Meenhard Herlyn (Wistar Institute, Philadelphia, PA) and maintained in a culture medium consisting of MCDB153 (Sigma Aldrich) with 20 % Leibovitz's L-15 (PAA Laboratories, Coelbe, Germany), 2 % FCS (PAA Laboratories), 1.68 mM CaCl₂ (Sigma Aldrich) and 5mg/ml insulin (Sigma Aldrich). Tissue origins of metastatic melanoma cell lines were the lymph nodes (WM239a, WM9, WM1158, and WM1232) and lungs (451Lu and 1205Lu). Primary melanocytes (NHEM) were isolated from human foreskins and purchased from PromoCell (Heidelberg, Germany) and cultured in Growth Medium M2 (C-24300).
Recombinant NRN1 was purchased from Sigma-Aldrich. Human NRN1 was expressed in E.coli and was diluted in PBS buffer containing 0.1 % BSA as a carrier protein as was used in a concentration of 50 ng/ml.

Melanoma cell lines Mel Im and Mel Juso were isolated from melanoma metastases and cultivated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with penicillin (400 units/mL), streptomycin (50 μg/mL), and 10% fetal calf serum (all from Sigma-Aldrich, Steinheim, Germany). WM3211 and SBcl2 were isolated from a primary tumor and cultivated in MCDB153 (Sigma-Aldrich, Steinheim, Germany) with 20% Leibovitz’s L-15 (PAA Laboratories, Coelbe, Germany), 2% FCS, 1.68 mM CaCl₂ (Sigma-Aldrich, Steinheim, Germany), and 5 µg/mL insulin (Sigma-Aldrich, Steinheim, Germany). All cell lines were incubated at 37 °C in a 5% CO₂, humified atmosphere [18 (link)]. All experiments were performed with mycoplasma-free cells (MycoSEQ mycoplasma detection system, Thermo Fisher Scientific, Waltham, MA, USA). Garcinol (Cay10566-5, Cayman Chemical, Biomol GmbH, Hamburg, Germany) treatment was performed with a stock solution of 5 mM dissolved in DMSO. Incubation time was 24 h. The final treatment concentrations are mentioned at the respective experiment or in the figure legend.