Sk mel 3

The cytotoxicity of Z1, Z2 and Z3 extracts was established by Neutral Red Uptake Test, as described by Repetto et al. [75 (link)] using human immortalized keratinocytes HaCaT (CLS Cell Lines Service GmbH, Eppelheim, Germany) [76 (link)], murine melanoma B16F10 (ATCC CRL-6475) and human melanoma A375 (ATCC CRL-1619) and SK-MEL3 (ATCC HTB-69) (LGC Standards, Łomianki, Poland). All cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM)/high glucose supplemented with 10% fetal bovine serum (FBS, Pan Biotech, Aidenbach, Germany) at 37 °C in a humidified atmosphere with 5% CO₂. For the experiments 3000 cells were plated per well onto a 96-well plate and grown overnight. Then, the cells were treated with various concentrations of Z1, Z2, or Z3 extracts (12.5–200 µg/mL) or an equal volume of the solvent control. Following 48 h of culture, the cells were incubated for 3 h in DMEM containing 1% FBS and 33 µg/mL neutral red, following by washing in PBS and lysis using acidified ethanol solution (50% v/v ethanol, 1% v/v acetic acid). The absorbance of the released neutral red was measured using FilterMax F5 microplate reader (Molecular Devices, San Jose, CA, USA) at λ= 540 nm. The mean measurement value for the lysate from control cells was set as 100% cellular viability and used to calculate the percentage of viable cells following extracts treatment.

HFF were purchased from ATCC (ATCC SCRC-1041). Three melanoma cell lines, Sk-mel-28 (ATCC HTB-72), Sk-mel-3 (ATCC HTB-69) and A375 (ATCC CRL-1619) were purchased from ATCC. All cell lines were cultured in DMEM (Gibco, 41966-029) supplemented with 10% FCS (Sigma Aldrich, F7524), 1× Glutamax (Gibco, 35050061), 100 U/ml penicillin and streptomycin (Gibco, 15140122) and 1 mM sodium pyruvate (Gibco, 11360070). Cells lines were cultured at 37 °C in 5% CO₂ with medium replaced as required. Cell lines were tested every fortnight for mycoplasma using LookOut Mycoplasma PCR Detection Kit (Sigma Aldrich, MP0035). Cell line identity was confirmed using STR profiling.

The experiments were performed in accordance with the Declaration of Helsinki guidelines. The study was approved by the Clinical Research Ethics Board of the University of British Columbia (Certificate H12-02653). With informed consent, biopsies were obtained and stored in RNAlater solution (Life Labs) as previously described [23 (link), 31 (link)] [32 (link)]. Biopsy tissues were archived and stored in −20°C in RNAlater solution (Invitrogen, Canada). Human epidermal melanocytes were purchased from ScienCell (Carlsbad, USA) and cultured in melanocyte medium (2201, ScienCell, Carlsbad, USA). Melanoma cell lines A375, RPMI 7951, SH4, WM-115, SK-MEL-1, SK-MEL-3, SK-MEL-24 were purchased from ATCC (Manassas, USA). Cells were cultured in growth medium as Dulbecco's modified Eagle's medium (DMEM) (Hyclone, Logan, USA) supplemented with 10% fetal bovine serum (Hyclone, Logan, USA) and 1X Antibiotic-Antimycotic (15240062, Gibco, Burlington, Canada) at 37°C in 5% CO2 humidified atmosphere.

Melanoma cell lines SK‐MEL‐3, SK‐MEL‐5, SK‐MEL‐28, Malme 3M and MeWo were purchased from ATCC and were grown using EMEM complete medium with non‐essential amino acids and pyruvate, or RPMI1640 medium (for Malme 3M). 501mel cells were generously provided by Dr. R. Halaban (Yale University) and maintained in RPMI1640 medium. All media were supplemented with 10% FCS and antibiotics. All cell lines harbour mutated BRAF(V600E), with the exception of MeWo cells which are BRAFwt; 293FT cells were purchased from Invitrogen (Carlsbad, CA, USA) and cultivated in DMEM with 10% FCS.