Fetal bovine serum (fbs)

Normal human epidermal keratinocytes (NHKCs; Kurabo, Osaka, Japan) were cultured in HuMedia-KG supplemented with insulin (10 μg/mL), hEGF (0.1 ng/mL), hydrocortisone (0.5 μg/mL), gentamicin (50 μg/mL), amphotericin B (50 μg/mL), and fetal bovine serum (0.4% v/v; Kurabo). Cells were cultured at 37°C in a 5% CO₂ atmosphere. Upon reaching 80% confluence, cells were treated with FICZ (10 nM), or DMBA (1 μM) for 4 h.

Mouse melanoma cells, B16F1 (ATCC CRL-6323), were grown on 10 cm dishes cultured in Dulbecco's Modified Eagle Medium high glucose (DMEM, Invitrogen, Gaithersburg, MD, USA) to which was added 10% fetal bovine serum (Invitrogen), 0.1 µg/mL streptomycin sulfate, and 100 U/mL potassium penicillin G (Invitrogen).
Human umbilical vein endothelial cells (HUVEC) were provided as frozen cells after primary culture by the supplier (Kurabo, Osaka, Japan). HUVEC were utilized as a model for human endothelial cells, and cultured on 10 cm dishes in HuMedia-EB2 (Kurabo, Osaka, Japan) consisting of 2% fetal bovine serum, 10 ng/ml human epidermal growth factor (hEGF), 1.34 µg/mL hydrocortisone hemisuccinate, 50 µg/mL Gentamicin, 50 ng/mL Amphotericin B, 5 ng/mL hEGF-B, and 10 µg/mL heparin, all supplied by Kurabo. Cells were cultured in a humidified 5% CO₂ incubator at 37°C.