Figure 3. Autophagy is a novel biological process regulating melanin production.

A) MNT-1 cells transfected with the indicated siRNAs (50 nM) were incubated in the presence and absence of bafilomycin A1 for 24 hours prior to lyses and analyses of tyrosinase protein accumulation. All results shown are representative of a minimum of three independent experiments. B) MNT-1 cells were transfected with the indicated siRNA pools (50 nM final concentration) or individual siRNAs (75 nM final concentration) targeting putative genes that regulate autophagy identified in the primary screen as described in Figure 1. WIPI1, LC3-C, and GPSM1 were genes identified in the original dataset, while BECN1 and MAP1LC3C are known regulators of autophagy not identified in our initial screening approach. Bars represent mean and s.e.m. for n = 3. Associated p-values (student's t-test) are reported in Table S4. C) Coat color defects in autophagy impaired mice. The coat pigmentation of C57B6 wild type (+/+) (mouse on the right) and heterozygous Beclin 1 mutant littermates (+/−) (mouse on the left) is shown. D) Reduced melanin accumulation in the hair follicles of Beclin1 haploinsufficient mice.. Skin samples from Beclin 1 haploinsufficient mice and wild type littermates were fixed and horizontally sectioned. Upper panels: Fontana-Masson silver staining was used to assess melanin content in the hair follicle (arrow). Detection of staining in wild-type follicles is obscured by accumulation of opaque pigment granules (left panel, and occasional normal follicles in the Beclin+/− background (arrow head)). Sections were counter-stained with aqueous neutral red. Lower panels: the melanoblast marker S100b was used to identify melanocytes in the hair follicle bulb (arrow). Again, staining is obscured in normal follicles due to accumulation of opaque pigment granules. E) MNT-1 cells were fixed and stained with the primary antibodies indicated. Two-photon confocal microscopy was utilized to visualize the colocalization of autophagy and melanosome markers. Representative 0.2 µM confocal slices are shown.