Synthetic melanin

The cells were lysed with 0.1 M phosphate buffer (pH 6.8) containing 1% Triton X-100 and protease inhibitor cocktail (Roche, Basel, Switzerland). The protein concentration in the supernatants was measured using Lowry assay system. Pellets were solubilized in 100 μL of 1 N sodium hydroxide (NaOH) for 3 h at 60 °C and the absorbance measured at 490 nm wavelength to analyze the melanin content. The melanin content was calculated from a standard curve using synthetic melanin (Sigma). For the assay of the tyrosinase activity, each sample was incubated with 2 mM L-DOPA (sigma) in 0.1 M phosphate buffer (pH 6.8) for 90 min at 37 °C. After incubation, the tyrosinase activity was measured at 490 nm wavelength.

Melanin from Oa1^−/− melanocytes expressing scrambled or OCA2-targeted siRNA was quantified as previously described (Oancea et al., 2009 (link)). Briefly, the soluble and insoluble fractions of melanocytes were separated after cell lysis with 1% Triton X-100 (Sigma) in PBS pH 7.4. Total protein was measured in the soluble fraction using a Bradford Assay (Bio-Rad Laboratories, Waltham, MA). The insoluble fraction was dissolved in 1 N NaOH by incubation for 30 min at 80°C and used to quantify melanin by measuring the optical density of each sample at 405 nm, then fit with a standard curve generated using synthetic melanin (Sigma). Average cellular melanin values were quantified as the ratio between total melanin and total protein from the same dish.

G361 melanoma (2 X 10³/well) and primary human melanocytes (5 X 10³/well) were plated in 96-well dish. Cells were treated with OAG typically for 24 h. For melanin assay, the cells were incubated with 0.85 N KOH (100 μl) with slow shaking at room temperature (RT) overnight. Melanin content was estimated by reading absorbance at 405 nm using a spectrophotometer (Tecan, Switzerland). Relative amount of melanin was calculated by using synthetic melanin (Sigma) as a standard in similar assays and normalized against protein content.

Quantification of melanin in iPSC-derived 3D skin equivalents was performed as described previously [27 ]. Briefly, frozen 3D skin constructs were thawed at room temperature and melanin was extracted using Solvable (PerkinElmer). A 1 mg/ml melanin standard stock solution was prepared by dissolving synthetic melanin (Sigma) in Solvable and a series of melanin standards were prepared from the stock. Solvable was used as the 0.0 mg/ml standard. The tissue samples and the melanin standard series were incubated for at least 16 hours at 60±2°C in a dry bath. The extracted tissue samples and the melanin standards were cooled and centrifuged. Samples or standards were then transferred to the appropriate wells of a 96-well plate (BD Biosciences), and Solvable was added to the wells designated as blanks. The absorbance at 490 nm of each well was measured with a Molecular Devices Vmax plate reader (Molecular Devices). The effect of forskolin on pigmentation in the 3D skin equivalents was determined in triplicate in 3 different experiments. Values are expressed as the mean % increase in melanin above control ± SEM. Statistical analysis was performed using the Student’s t test and significance level has been identified as p<0.05.

Cells were transfected and seeded at a density of 1x10⁵ cells/ml in 6-well plates. 72h post transfection, cells were harvested, washed in PBS and lysed in 1N NaOH in 10% DMSO. Cellular melanin was dissolved by incubating the homogenate at 80°C for 2h and then centrifuged at 500xg for 5 min. Absorbance of the supernatant was measured at 405nm. Synthetic melanin (Sigma) was used as standard, ranging from 0–80 μg/ml.

Murine melanoma B16-F1 cell line (CRL-6323) was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured in Dulbecco’s modified Eagle’s Medium (DMEM; Nacalai Tesque Inc., Kyoto, Japan) supplemented with 10% (v/v) foetal bovine serum (HyClone Laboratories, Logan, UT, USA) and 1% (v/v) penicillin G (100 U/mL) and streptomycin (100 µg/mL) (gibco, Thermo Scientific, Waltham, MA, USA). The cells were grown and maintained at 37 °C with 5% CO₂ and humidity. Monoclonal antibodies against phospho-p38, total p38, and cleaved caspase 3 were obtained from Cell Signaling Technology (Danvers, MA, USA). A monoclonal antibody against β-actin was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Phosphate buffered saline (PBS), 0.05% trypsin, and phenol red-free DMEM were purchased from Gibco (Thermo Scientific). Dimethyl sulfoxide (DMSO) was purchased from Kanto Chemical Co. (Tokyo, Japan). Methanol and absolute ethanol were purchased from Fisher Scientific (Thermo Scientific). MTT, αTP, synthetic melanin, l-3,4-dihydroxyphenylalanine (L-DOPA), bovine serum albumin (BSA), DCFDA, and JC-1 kit were obtained from Sigma-Aldrich (St. Louis, MO, USA). TRF with a purity of ≥95% was supplied by Davos Life Science Sdn Bhd (DavosLife E3, Petaling Jaya, Malaysia).

Dulbecco’s phosphate-buffered saline (DPBS), neutral red solution (3.3 g/L in DPBS), Dulbecco’s modified Eagle’s medium (DMEM), Eagle’s minimum essential medium (EMEM), synthetic melanin, glucose, L-3,4-dihydroxyphenylalanine (L-DOPA), murine tyrosinase, kojic acid (≥98.5%) were acquired from Sigma Aldrich/Merck (Darmstadt, Germany). Fetal bovine serum (FBS) was from Pan Biotech (Aidebach, Germany), whereas McCoy’s 5a medium was from LGC Standards (Łomianki, Poland). Ethanol, acetic acid, and sodium hydroxide (NaOH) were purchased from Honeywell (Charlotte, NC, USA). Cannabidiol (CBD, >99.0%), cannabigerol (CBN, >98.0%), cannabinol (CBN, >98.5%), and cannabichromene (CBC, >95.0%) were isolated from hemp flower extracts, as described in [32 (link),33 (link)].