Melanin content measurement
B16 cells were seeded in a 6-well plate at a density of 1.8 ×
105 cells/well. After 24 h of incubation, the cells
were treated with different concentrations of the test compounds for 48
h, 8-MOP was added at 50 µM as a positive control, and 2 µL of DMSO was
added to the control group. Subsequently, B16 cells were washed twice
with PBS (pH 7.4) and lysed in 100 μL of RIPA lysis buffer (AR0105-100)
(BOSTER Biological Technology, Wuhan, China) for 40 min at 4 °C. The
lysates were centrifuged at 12,000 × g for 20 min at 4 °C. We collected
3 µL of supernatant from the cell extracts to measure the total protein
content using a BCA kit assay (PP02) (Biomed, Beijing, China). The
pellets were treated with 190 μL of 1 M NaOH (with 10% DMSO) for 1 h at
80 °C, and the optical density was detected at 405 nm using a
multi-plate reader (SpectraMax M5/M5e). The melanin content was
calculated relative to that in the control group and corrected for the
protein concentrations, considering the control group as representing
100% melanin content.