Fig. 3

Autophagy was required for the anti-melanogenesis effect of hAMSC-CM or hAESC-CM. B16F10 cells were exposed to hAMSC-CM or hAESC-CM and then the lysates were collected for western-blotting analysis using LC3- and p62-specific antibodies and β-Actin was used as a loading control (a). B16F10 cells stably expressing dual-fluorescence mRFP-GFP-LC3B were treated with hAMSC-CM or hAESC-CM for 48 h and then analyzed by fluorescence microscopy, and the red and yellow puncta were counted under 40 × magnification. Scale bar = 20 μm (b). B16F10 cells stimulated with α-MSH were incubated with hAMSC-CM or hAESC-CM. The melanin content was analyzed by measuring absorbance in the presence or absence of bafilomycin A1 (c). B16F10 cells pre-treated with α-MSH were exposed to hAMSC-CM or hAESC-CM for 48 h and observed with electron microscopy (scale bar = 2 μm), and the red arrows indicate over-produced melanin (only α-MSH group) and autophagosomes that contain melanin or melanosome (hAMSC-CM or hAESC-CM groups) in the higher magnification electron microscopic picture (scale bar = 500 nm) (d). Melanosomes were added to the HaCat cells, followed by incubation for 24 h. After washing with PBS, the cells were incubated with hAMSC-CM or hAESC-CM for an additional 48 h, then the cells were collected and lysed for melanin content assay (e) and anti-LC3B, anti-P62 antibodies (f). The protein intensity was calculated from normalizing against β-actin. Data are presented as mean ± SD. The experiments were repeated three times independently and the data of one representative experiment was shown. *p < 0.05; **p < 0.01, ***p < 0.001