Hemn mp

Four melanoma cell lines were supplied by Prof Xu Dong Zhang: MM200, Sk-mel-28 Mel-RM and Me4405. The tumour status [40 (link), 41 (link)] and p53 status [42 (link)] of each melanoma cell lines have been previously described. Human neonatal, medium (HEM1455 and HEMn-MP) and dark (HEMn-DP) pigmented epidermal melanocyte cell lines were purchased (Cascade Biologics, USA and ThermoFisher, USA). Cell line authentication was performed as previously described [16 (link)] and using GenePrint 10 (Promega, USA). Mycoplasma was tested and not detected at 6 month intervals using the MycoSEQ mycoplasma detection kit (Life Technologies, USA). Melanoma cell lines were cultured in 1x DMEM (Gibco, Life Technologies, USA) and melanocytes were cultured in Medium 254 (Gibco, USA) All cells were incubated at 37°C 5% CO₂.

Murine melanoma B16F10 melanoma cells were obtained from the Korean Cell Line Bank (Seoul, Korea) and cultured in phenol red-free DMEM supplemented with 10% FBS and 1% penicillin/streptomycin in a humidified atmosphere containing 5% CO₂ at 37 °C and sub-cultured every 96 h. Cells were treated with PH for 72 h (for tyrosinase activity and melanin content assay), and α-MSH (200 nM) and arbutin (100 μM) were used as negative and positive controls, respectively. Human epidermal melanocytes (moderately pigmented donor, HEMn-MP) were purchased from Life Technologies (Carlsbad, CA, USA). The cells were cultured in 254 medium (Life Technologies) supplemented with 1% human melanocyte growth supplements and 1% penicillin/streptomycin in 5% CO₂ at 37 °C.

Patient derived cell lines were provided by the University of Colorado Skin Cancer Biorepository (patient consent and specimen usage outlined under COMIRB 05-0309) and validated by melanoma triple cocktail staining. Patient lines were derived from metastases of patients seen at our institution, and include samples derived from patients relapsed from current treatments. Patient lines were Short tandem repeat (STR) profiled with >80% match to the patient’s corresponding tumor or blood sample. Genetic backgrounds are listed in Table S3. All cell lines were maintained in RPMI 1640 medium (Invitrogen, Grand Island, NY, USA) with 10% fetal bovine serum (Gemini Bio-Products, Inc., West Sacramento, CA, USA) and were tested for mycoplasma. Primary melanocytes HEM_NMP were obtained from Life Technologies (Carlsbad, CA, USA). Melanocytes were maintained in Medium 254 with Human Melanocyte Growth Supplement-2 (Life Technologies, Carlsbad, CA, USA). To mimic melanoma culture conditions, 10% FBS was added for drug assays.