Kuge et al., "The expression of tyrosinase, tyrosinase-related proteins 1 and 2 (TRP1 and TRP2), the silver protein, and a melanogenic inhibitor in human melanoma cells of differing melanogenic activities," Pigment Cell Research, vol.
Results showed that the mRNA levels of Tyr and Trp1 genes were significantly increased, 4 h after treatment with CYM or [alpha]-MSH compared to the control (Figures 3(a) and 3(b)) while Dct gene expression was only slightly increased (5% increase compared to the control) (Figure 3(c)).
(Table 1) showed that CYM upregulated the expression of the three melanogenic enzymes Tyr, Trp1, and Dct as well as cytoskeleton organization-associated genes, copper transport and most melanosome transport genes such as thymosinbeta 10 (TMSB10) and ribosomal protein S3 (Rps3).
To determine if the increase in melanogenesis was caused by an increase in the melanogenic enzymes tyrosinase (TYR), tyrosinase-related protein 1 (TRP1), or dopachrome tautomerase (DCT) expression, total protein of B16 cells treated with or without 1/1,000 v/v CYM or [alpha]-MSH was extracted and western blotting was carried out.
Like in B16 cells, the expression of TYR, TRP1, and DCT was also increased by CYM.
Primers specific to tyrosinase (Tyr), tyrosinase-related protein 1 (Trp1), dopachrome tautomerase (Dct), and microphthalmia-associated transcription factor (Mitf) were used for quantitative real-time PCR performed with a 7500 Fast Real-time PCR system using TaqMan Universal PCR mix and TaqMan probes (Applied Biosystems, Foster City, CA, USA).
Protein samples (10 [micro]g) were resolved in 10% sodium dodecyl sulphate-polyacrylamide gel by electrophoresis (SDS-PAGE), transferred to PVDF membrane (Merck Millipore, USA), and blotted with primary antibodies for tyrosinase (TYR), tyrosinase-related protein 1 (TRP1), dopachrome tautomerase (DCT), and GAPDH.
In pigment cells, oxidative stress causes a downregulation of the melanogenic enzymes TYR, TRP1, and DCT and their transcription factor MITF, leading to decreased melanin biosynthesis [14,15].
We further tested the protein expression of TRP1, TRP2, tyrosinase, and [beta]-catenin in different experimental groups by western blots.
To investigate the effect of sFRP4 on the differentiation of McSCs/melanocytes, we examined the expression of TRP1, which is a differentiation marker of McSCs/melanocytes .
We then performed double immunofluorescent staining for sFRP4 and TRP1 (a marker of melanocytes)  to determine whether sFRP4 is expressed in follicular melanocytes.
After blocking, sections were incubated with the following primary antibodies: goat anti-sFRP4 (1: 100, Abcam, Cambridge, USA), goat anti-TRP2 (1:100, Santa Cruz, City of Santa Cruz, CA, USA), mouse anti-AE13 (1: 100, Santa Cruz, City of Santa Cruz, CA, USA), mouse anti-AE15 (1:100, Abcam, Cambridge, USA), rabbit anti- TRP1 (1: 100, Santa Cruz, City of Santa Cruz, CA, USA), rabbit anti-[beta]-catenin (1:200, Abcam, Cambridge, USA; 1:100, Beyotime, Beijing, China), and rabbit anti-PCNA (1: 100, Abcam, Cambridge, USA).