Fig 2: Signaling pathway changes after narrow-band ultraviolet B (NB-UVB) exposure. (A) Effect of mRNA expression in differentiated melanocytes from HS-NCSCs after NB-UVB radiation. (B) Western blot analysis of Tyrosinase and Mc1R protein levels after NB-UVB radiation. H89 and PD98059 were added in the melanocyte culture medium, respectively. Molecular weight of Tyrosinase is 60 kD, Mc1R is 35kD and ß-actin is 45kD. HS-NCSCs: human scalp-derived neural crest stem cells, Mc1R: Melanocortin 1 receptor, Tyro: tyrosinase. *p<0.05, compared with control.

Fig 3: Targeted inhibition of APT2 inhibits ultraviolet B (UVB)-induced melanomagenesis in vivo. a Illustration of the dynamic melanocortin-1 receptor (MC1R) palmitoylation/depalmitoylation cycle. b cAMP levels in human primary melanocytes (HPMs) after treatment with increasing concentrations of the indicated inhibitors. HPMs in which endogenous MC1R is stably depleted using shMC1R were infected with Flag-MC1RR151C and then treated with 1 µM a-melanocyte-stimulating hormone (a-MSH) and indicated inhibitors for 3.5 h. The resulting cells were harvested for a cAMP immunoassay. The data were compiled from five independent experiments. c MC1R-depleted HPMs were infected with the indicated Flag-MC1R-encoding retroviruses and then pretreated with 1 µM a-MSH and 100 nM ML349 for 30 min followed by 100 J/m2 UVB irradiation. The resulted cells were harvested for immunoprecipitation, acyl-biotin exchange, and immunoblotting analysis with the specific antibodies as indicated 3 h after UVB exposure. d cAMP levels in MC1R-depleted HPMs expressing the indicated Flag-MC1R encoding retroviral constructs and pretreated with 1 µM a-MSH and 100 nM inhibitors for 30 min followed by 100 J/m2 UVB irradiation. The resulted cells were harvested for cAMP immunoassay 3 h after UVB exposure. Data were compiled from five independent experiments. e–g Growth curves (e), dissected tumors (f), and tumor weight (g) for the xenograft experiments. MC1R-depleted hTERT/p53DD/CDK4(R24C)/BRAFV600E melanocytes were infected with the indicated Flag-MC1R encoding retroviral constructs. Cells were preincubated with 1 µM a-MSH and 100 nM inhibitors for 30 min before being irradiated with 20 J/m2 UVB. After 24 h, 3 × 106 cells were inoculated subcutaneously into each flank of nude mice. Visible tumors were measured at the indicated days. h Melanoma-free survival of the indicated mice. In the UVB radiation period, 5 mg/kg ML349 was injected intraperitoneally into mice prior to the treatment with UV. Ninety days after the final UVR, melanoma was diagnosed in 23% (3/13) or 64% (7/11) of mice with or without ML349 treatment, respectively (p = 0.0366). **p < 0.01, ***p < 0.001, unpaired Student’s t test. Error bars represent ± s.d.