B16f10 cells

B16F10 murine and SK-MEL-28 human melanoma cell lineswere purchased from the Korean Cell Line Bank (Seoul, Korea). The cells were cultured in Dulbecco's modified Eagle's medium (B16F10 cells) or modified Eagle's medium (SK-MEL-29 cells, Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Atlas Biologicals, For Collins, Co, USA), 100 U/ml penicillin and 100 μg streptomycin (Sigma-Aldrich). Human umbilical vein endothelial cells (HUVECs) and their growth medium (Endothelial Cell Growth Medium) were purchased from PromoCell GmbH (Heidelberg, Germany). The cells were used at passage 4-5 in all experiments. All cells were incubated at 37 °C with 5% CO₂. Naringenin was dissolved in dimethyl sulfoxide to obtain a 200 μM stock solution, which was then diluted with media.

Eight-ten week old C57BL/6 mice (Charles River Canada, Saint-Constant, Canada) were used to generate a melanoma mouse model. Animals were housed under conventional conditions at the Animal Care Facility, Western University, and were cared for in accord with guidelines established by the Canadian Council on Animal Care. Tumors were generated on the backs of each mouse by subcutaneous injection of 4x10⁵ B16F10 cells in PBS (Gibco). Tumors were allowed to grow for 16 days or until reaching a size of 2,000 mm³. Mice were monitored daily and euthanized by CO₂ inhalation.

Mouse melanoma B16F10 cells and human dermal fibroblasts (HDFns) were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). B16F10 cells were cultured in Dulbecco’s modified eagle medium (Gibco, Paisley, UK) supplemented with 10% fetal bovine serum (Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin. The HDFn cells were cultured in F12: Dulbecco’s modified eagle medium (Gibco) supplemented with FBS and penicillin/streptomycin. The cells were subcultured every 2 to 3 days at 37 °C in a 5% CO₂ incubator.

The B16F10 cells and HepG2 cells were purchased from the National Collection of Authenticated Cell Cultures and cultured in Dulbecco’s modified eagle medium (DMEM; Gibco, Beijing, China), which was supplemented with 10% newborn bovine serum (NBS; Every Green, Huzhou, China) and 1% penicillin–streptomycin (Sangong Biotech, Shanghai, China) and incubated at 37 °C, 5% CO₂.
The B16F10 cells and HepG2 cells were planted separately in 96-well plates with a density of 7000 cells per well and incubated for 12 h. Then, different groups (Quercetin, QC and QCD) were added to the fresh, complete medium according to the gradient concentrations of 4, 8, 16, 32 and 50 μg/mL. The sample-free cell culture plate was used as a control. After 24 h of incubation, the medium was removed and 100 μL of MTT solution (0.5 mg/mL) was added to each well for another 4 h incubation. Then, the MTT solution was replaced with 100 μL of DMSO. After 15 min in a shaker at 37 °C, the absorbance at 490 nm was read by a multifunctional enzyme labeling instrument (Synergy H4, Bio-Tek, Winooski, VT, USA). For the QCDA group, all procedures were the same as above except for an additional 5 min of laser irradiation exposure (808 nm, 1.5 Wcm⁻²).

Human HaCaT cells, HDFs, NHEKs, NHEMs and murine B16F10 cells were purchased from Thermo Fisher Scientific (Waltham, MA, USA). HaCaT cells, HDFs and B16F10 cells were cultured in Dulbecco’s modified Eagle’s medium (HyClone, GE Healthcare Life Sciences, Logan, UT, USA) with 10% fetal bovine serum (HyClone) and 1% penicillin–streptomycin (LS202-02, Welgene, Daegu, Korea). NHEKs and NHEMs were cultured in keratinocyte media (MEP1500CA, Gibco, Life Technologies, Carlsbad, CA, USA) and Medium 254 (M254500, Gibco) supplemented with human keratinocyte and melanocyte growth supplement (S0015, S0025, Gibco) and 1% penicillin-streptomycin at 37 °C in a humidified mixture of air (95% (v/v)) and 5% CO₂. For LTAP exposure, 5 × 10⁵ cells were seeded in 35 mm dishes and incubated for 24 h. Before LTAP exposure, the medium was exchanged for 2 mL of new growth medium. The lid was opened, and the dish was placed 1 cm below the part where argon gas was ejected from the LTAP generator, and exposed for 1, 3, or 5 min. After exposure for the indicated time, the dishes were placed in a CO₂ incubator at 37 °C.

Human newborn foreskin (BJ) fibroblasts and B16-F10 mouse skin melanoma cells were purchased from the American Tissue Culture Collection (ATCC), while the human neonatal foreskin fibroblast strain AG01523 was obtained from the Coriell Institute for Medical Research. BJ cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 10% (v/v) fetal bovine serum (FBS), 2 mM glutamine and 1% non-essential amino acids, whereas AG01523 cells were cultured in DMEM containing 15% (v/v) FBS and 2 mM glutamine, and B16-F10 cells in DMEM containing 10% (v/v) FBS and 2 mM glutamine. All cells were maintained in a humidified environment of 5% CO₂ and 37 °C. They were subcultured using a trypsin-citrate solution (0.25–0.3%, respectively), and they were tested periodically and found to be mycoplasma free.

C57BL/6 female mice (6–9 weeks old) were injected intra-peritoneum with PBS containing 3 × 10⁶ B16-F10 cells (BNIP3^WT or BNIP3^KD) pHrodo-labelled (Life Technologies, P36600). The peritoneal cells were collected 24 h post-injection via peritoneal lavage with Ca²⁺- and Mg²⁺-free PBS. The cells were transferred to an ultra-low attachment V-bottom plate and stained with the fixable Live/Dead Yellow stain (Invitrogen). After blocking the F_c receptor (CD16/32, eBioscience, 16-0161-82), cells were stained for F4/80-eFluor780 (eBioscience, 47-4801-80), CD11b-eFluor450 (eBioscience, 48-0112-82), MHCII-FITC (eBioscience, 11-5321-81), CD86-APC (eBioscience, 17-0862-81), CD206-PeCy7 (eBioscience, 25-2061-80) in FC buffer. Samples were acquired on a Gallios^™ flow cytometer and the data analysis was performed via FlowJo_V10^™ software.