Time course of treatment of melanocytes with ASP, PTU or αMSH. (top left) Experimental design: melanocytes were seeded 2 days prior (−2D) to the first treatment with 10 nM ASP, 200 μM PTU or 100 nM αMSH in fresh media (D0) to start the 4 days of treatment. At D2, media were collected and replaced with fresh media containing the drugs or vehicle as appropriate (4 and 2 days of treatment). At D3, appropriate drugs or vehicle were added to the remaining dishes for 1 day of treatment. Cells were collected at D4, frozen and lyophilized, while media were combined with those collected at D2, prior to lyophilization. Bright field microscopy of cells before harvesting of each treatment as noted (all photos at the same 10X original magnification and exposure settings); insets show representative cells at higher magnification (each at 40X) and the visual appearance of each cell pellet after harvesting.