Figure 1 . Identification of new phenanthridine Wnt/β -catenin signalling agonists, HCJA121 and HCJA404, and their effects on melanogenesis in B16-F10 cells. (a) Structures of HCJA121 and HCJA404. (b) The levels of melanin in phenanthridine-treated B16-F10 cells. B16 cells were seeded in a 6-well plate and incubated with Wnt3a before being treated with 50 µ M of HCJA121, HCJA404, and compounds 16 for 48 h; 8-MOP served as a positive control and DMSO as blank control. The optical density was detected at 405 nm. The melanin content was calculated relative to that in the control group and was corrected on the basis of the protein concentrations, considering the control group as representing 100% melanin content. (c) HCJA121 and HCJA404 (20 μ M) significantly increased tyrosinase activity in B16-F10 cells. The cellular tyrosinase activity was estimated by measuring the rate of ʟ-3,4-dihydroxyphenylalanine (ʟ-DOPA) oxidase activity. The tyrosinase activity of each sample was calculated and corrected for the concentrations of proteins, considering the control group as representing 100% activity. (d) The MITF regulatory network underlying melanogenesis. (e) HCJA121 and HCJA404 increased the expression of the MITF , TYR , and TYRP 1 and2 genes. B16 cells pre-treated with HCJA121, HCJA404, or 8-MOP for 2 h were incubated with Wnt3a-conditioned medium containing the same dose of compounds for an additional 48 h. MITF , TYR , andTYRP 1 and 2 were analysed by qPCR. The results were normalized to β -actin expression. (f) Compound HCJA121 increases the levels of the MITF, TYR TYRP 1, and TYR TYRP 2 proteins. B16 cells treated with HCJA121 for 48 h were fractionated as described in the Methods section and subjected to western blot analysis. β -actin served as the loading control. The immunoblots were quantified by densitometry, and the intensity values were normalized toβ -actin. The values are indicated beneath each band. The data shown represent means ± standard deviations (S.E.M) from five independent experiments (n=5). Significantly different as indicated (*P < 0.05, **P < 0.01, ***P< 0.001, and ****P < 0.0001).