Penicillin streptomycin solution

Kaempferol, AAPH, superoxide dismutase (SOD), malondialdehyde (MDA), and cytotoxicity assay (CCK-8) kits were obtained from Solarbio (Beijing, China). 3,5-dimethylphenol was obtained from Life Technologies (Carlsbad, CA, United States). Phosphate-buffered saline (PBS) was procured from Biological Industries (Ridgefield, CT, United States). Penicillin–streptomycin solution was obtained from ScienCell (San Diego, CA, United States). Dulbecco’s modified Eagle’s medium (DMEM) and trypticase were purchased from HyClone (Logan, UT, United States). Fetal bovine serum (FBS) was procured from Gibco (Waltham, MA, United States). A BCA Protein Quantitation Kit was obtained from Thermo Fisher Scientific (Waltham, MA, United States).

The human hepatoma HepG2 cell line (ATCC-HB-8065) was cultured in EMEN medium (Quality Biological, Gaithersburg, MD, USA) supplemented with 10% FBS (Biowest, Riverside, MO, USA) and 1% penicillin/streptomycin (Sigma-Aldrich, St. Louis, MI, USA). The cells were maintained in a humidified atmosphere with 5% CO₂ at 37 °C [36 (link)]. Primary HEKn cells were cultured in KM culture medium (ScienCell Research Laboratories, Carlsbad, CA, USA) supplemented with KGS 100× (ScienCell Research Laboratories, Carlsbad, CA, USA) and 1% penicillin/streptomycin solution (ScienCell Research Laboratories, Carlsbad, CA, USA). All experiments were conducted using cells in the logarithmic growth phase.

Melanocyte culture was established using the method described by Chen et al. [22 (link)]. An adult black rabbit was used to isolate the melanocytes. A tissue sample of approximately 1.5 cm × 1.5 cm was excised from the dorsal skin of the rabbit. Rabbit melanocytes were cultured in Melanocyte Medium-2 (ScienCell, San Diego, CA, USA) supplemented with 1% PMA-free melanocyte growth supplement, 0.5% fetal bovine serum (FBS), and 0.5% penicillin/streptomycin solution (ScienCell, San Diego, CA, USA). Melanocytes were transferred to 24-well plates and incubated at 37 °C with 5% CO₂. When the culture was 70–90% confluent, overexpression and knockdown experiments were performed using 1 μg GNAI2 plasmid and 1 μL siRNAs (Table 2), respectively. Both were diluted in 25 μL Opti-MEM™ medium (Gibco, Carlsbad, CA, USA) and 2 μL diluted Lipofectamine™ 3000 (Lipofectamine™ 3000 diluted in 25 μL Opti-MEM™ medium) was added to both the mixtures. These mixtures were incubated for 10-15 min at room temperature and were then added to cultured melanocytes separately, and the cells were incubated at 37 °C with 5% CO₂ for 48 h.

Human PMVEC were cultured as previously described (Pool et al., 2018 (link); Toby et al., 2010 (link); White et al., 2017 (link)). Briefly, fetal hPMVEC were purchased from ScienCell (Carlsbad, CA) and were all from females including lot#5016, lot#15900, lot#17807, and lot#15902. The hPMVEC were cultured in endothelial cell (EC) media (ScienCell) supplemented with an EC media kit containing fetal bovine serum, endothelial cell growth supplement, and a penicillin/streptomycin solution (ScienCell). Human PMVEC between passages 4 and 7 were used to generate cultured media. The hPMVEC were incubated at 37°C in either 5% CO₂, balance air (normoxia) or 5% CO₂, 1% O₂, balance N₂ (hypoxia) for 24 h, when the media was harvested for the conditioned media experiments. The media from the hPVMEC incubated in normoxia was termed normoxic conditioned media (NCM) and the media from the hPMVEC incubated in hypoxia was termed hypoxic conditioned media (HCM). For some experiments, 1 µg/mL of EGF neutralizing antibody (anti‐hEGF; R&D Systems, Minneapolis, MN) or an IgG control antibody was added to the EC media prior to generating HCM (EGFnAb‐HCM or IgG‐HCM). For other experiments, 20 μg/mL of EGF receptor blocking antibody (anti‐EGFR; EMD Millipore, Temecula, CA) or an IgG control antibody were added to the EC media prior to generating HCM (EGFRbAb‐HCM or IgG‐HCM).