Figure 1.
Haematoxylin and eosin staining of mouse skin of different age and hair cycle.
(A,C) C57BL/6. (B,D) C57BL/6-HGF/SF. (A-B) PND21 (postnatal day 21). (C-D)- PND5 (postnatal day 5). Arrows – extrafollicular melanocytes; T- telogen hair follicle; s 7/8- stage 7/8 of hair follicle morphogenesis. Scale bar - 100 µm.
Figure 2.
TEM pictures of melanocytes in mouse hair and skin.
(A-B) Melanocytes in adult hair shaft; (A) wild-type C57BL/6 (B) C57BL/6-HGF/SF. (C-F) Melanocytes in C57BL/6-HGF/SF transgenic mouse; (C) hair bulb. (D-E) adult skin. (F). PND5 skin. f- fiber, n-nucleus, l- lamellar structure. Melanosome differentiation stages I-IV.
Figure 3.
EPR spectra of the investigated materials.
DPPH (1,1-diphenyl-2-picrylhydrazyl) – marker for the position of the free radical signal (g=2.0037).
(A) Dopa melanin and cysteinyl dopa melanin. (B) Hair of adult mouse (20 mg). (C) Melanin in skin (100 mg) of adult. (D). PND5 mouse skin. *- indicates the low field component of the splitting, used to calculate the a/b parameter (A). C57BL/6 and C57BL/6-HGF skin is compared to samples known for its content of pheomelanin, and often used as a pheomelanotic standard: hair of yellow C57BL/6-Ay/a and C57BL/6- Mc1re/e mice, and with amelanotic C57BL/6-c. Note that the pigment contained in the skin and in the hair is not pure pheomelanin, but a co-polymer with high contribution of pheomelanin. Also the used synthetic cysteinyldopa-melanin, besides semiquinonimine centres, contains semiquinones typical of eumelanins, with no interacting 14N.
Figure 4.
Quantitative analysis of the EPR measurements.
(A-B) The EPR signals line widths of analyzed materials. (C-D) Amplitudes of the EPR signals normalized per tissue sample mass and amplification. (E) Skin thickness (mm). (F-G) Normalized amount of melanin per skin thickness. h- normalized amount of melanin per skin thickness and number of hair follicle (#HF) count per microscopic field 10×(MF). All the bars represent the means of 3-4 samples ± SD.
Figure 5.
Tyrosine activity and expression of tyrosinase, Dct and MITF in PND5 and 3 weeks (adult) C57BL/6 mouse skin.
(A) Zymography of tyrosinase activity. (B) Densimetric analysis of the zymogram, positive control-purified tyrosinase from Agaricus bisposrus, 5 mU (TYR 5 mU), negative control C57BL/6-c. (C) Immunoblotting. (D) Densitometric analysis of the bands – Tyrosinase (Tyr), DOPAchrome tautomerase (Dct) and MITF expression was normalized to a reference protein Grb-2, that served as a loading control. The Western blot experiments were performed on two different set of the samples, each set repeated twice. The figure shows the representative results.