Fig 1: MCs achieve the phenotypes of melanogenesis and migration following UVB exposure. Primary human epidermal MCs were isolated from juvenile foreskin tissues, treated with or without 30 mJ/cm2 UVB and then cultured for one additional day. (a) qRT-PCR was performed to measure the expression levels of melanogenesis-associated mRNAs (MITF, TYR, TYRP1, DCT, and Pmel17) and the migration-related MCAM in MCs exposed or unexposed to 30 mJ/cm2 UVB. The results were normalized to the housekeeping gene GAPDH. The data represent means ± SD from 3 independent experiments. *P < 0.05; **P < 0.01. (b) MCs were lysed in extraction buffer, after which specific antibodies against human MITF, TYR, TYRP1, DCT, Pmel17, and MCAM were used in western blotting to examine their corresponding protein levels. Protein loading variations were determined by immunoblotting with an anti-GAPDH antibody. Representative blots are shown on the left. The histograms show the densitometric quantification of data with means ± SD from 3 independent experiments. *P < 0.05; **P < 0.01. (c) Supernatants were collected from UVB-exposed and from unexposed MCs, and the protein concentration in each supernatant was measured using the BCA assay, after which the supernatants were further concentrated by lyophilization as described in Materials and Methods. Levels of sPmel17 in the supernatants from UVB-exposed or UVB-unexposed MCs detected by western blotting are shown (upper panel, right) (**P < 0.01). An equal amount of total protein (20 µg) of each supernatant was loaded per lane and was separated by SDS-PAGE. A gel stained with Coomassie blue solution is shown (left panel). The black arrowhead indicates the band at 100 kDa of presumed sPmel17. (d) Representative images for cell migration assessed by the Transwell assay (scale bars = 20 µm). Histograms show the number of cells passing through the insert membrane. The data represent means ± SD from 3 independent experiments. **P < 0.01.
Fig 2: Saponins-rich fraction of argan leaves extract (ALS) modulates melanogenic enzymes expression in B16 cells. B16 cells were seeded at a density of 5 × 105 cells per 100 mm Petri dish. After overnight incubation, cells were treated with 10 µg/mL of saponins-rich argan leaf sample (ALS) or 100 µM arbutin (Arb) then incubated further at 37 °C for 24 h or 48 h prior to protein extraction. Protein expression was determined by Western blotting. (A) Protein expression of melanogenesis enzymes tyrosinase (TYR), tyrosinase-related protein 1 (TRP1), and dopachrome tautomerase (DCT). (B) Protein expression of microphthalmia-associated transcription factor (MITF); gene expression of melanogenesis enzymes tyrosinase (Tyr) (C), tyrosinase-related protein 1 (Trp1) (D), and dopachrome tautomerase (Dct) (E) determined using TaqMan real-time PCR.). * indicates significance at p < 0.05.
Fig 3: Analysis of hair follicloids and generated hair shafts.(A) Hair follicloids after 10 days of culture. Whole-mounted hair follicloids were stained with 4′,6-diamidino-2-phenylindole (DAPI) and observed using a confocal laser microscope (i) and scanning electron microscope (SEM) (ii). The whole-mounted hair follicloids were stained with oil red O and observed using a stereomicroscope (iv). The hair follicloids were sectioned and stained with HE (iii), a fluorescent antibody against α-SMA (v), Sox9 (vi), CD34 (vi), K5 (vii), Ki67 (viii), versican (ix), gp100 (x), TRP1 (xi), and c-Kit (xii). The inserted images show magnified views of the box area in the images. BF, bright field. (B) Hair cuticles of generated hair shafts. The hair shaft was observed after 10 days of culture using a scanning electron microscope. (C) Hair bulb of generated hair follicle after 10 days of culture. Hair follicloids were observed using a stereomicroscope. (D) Microstructures of generated and native hair shafts. Cross sections were observed via transmission electron microscopy. White arrowheads indicate melanin granules, and black arrowheads indicate hair cuticles.
Fig 4: Effects of THC on mRNA expression of genes for TYR (A), TRP-1 (B), and TRP-2 (C) in B16F10 melanoma cells. Levels of mRNA were determined by PCR and GAPDH was used as the internal reference. Results are expressed as mean SD (n = 3). *** p < 0.001 compared to group treated with a-MSH.
Fig 5: SKL2001 reverses the inhibitory effect of SFRP5 on melanogenesis. (A) The optimal concentration of SKL2001 was detected by Western blot. (B) Western blot analysis of protein expression levels of SFRP5, MITF, TYR, TRP1 and TRP2 in PIG1 cells. (C) Results of tyrosinase activity measurement. (D) Results of melanin content measurement. Error bars are the means ± SD, n = 3. ***P < 0.001, **P < 0.01 or *P < 0.05 vs. control.
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