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Fig. 10 3-MA negatively affected the anti-melanogenic activity of Pt in B16F10 cells. (A?B) Cells were first treated or not with 3-MA (1 mM, 1 h) followed by Pt (0?30 ?M) for 24 h (A) or 72 h (B). After treatments, an MTT assay was performed to determine the cell viability. (C?E) Cells were first treated or not with 3-MA (1 mM, 1 h), followed by 30 ?M Pt treatment for different time points to allow the expression of various melanogenesis-associated proteins - p-CREB (2 h), CREB (2 h) (C), p-MITF (4 h), MITF (4 h) (D), tyrosinase (24 h), TRP-1 (24 h), TRP-2 (24 h) (E) through the Western blot method. ?-actin functioned as a loading control. (F) Cells were treated with Pt (30 ?M) in the absence or presence of 3-MA (1 mM, 1 h) followed by stimulation with ??MSH (1 ?M, 24 h). The melanosome-engulfing autophagosomes in the cells were analyzed through transmission electron microscopy (Tecnai 12, FEI, Hillsboro, Oregon USA) (Bar = 1 ?m). The black arrows indicate autophagosome containing melanin or melanosome. (G) Cells were first treated or not with 3-MA (1 mM, 1 h), and then with Pt (30 ?M, 72 h) followed by stimulation with ??MSH (1 ?M, 24 h). Cells were harvested, and intracellular melanin levels were estimated as described in the methodology. Cells treated with 30 ?M Pt alone or ??MSH alone or 3-MA alone functioned as controls. Results were denoted as mean ± SD of three or more independent experiments. Statistical significance was considered as ***p < 0.001 compared to untreated control cells or ??MSH-stimulated cells. ##p < 0.01 compared to ??MSH + Pt treated cells.