Fig. 8. Downregulation of Mitf mRNA levels in B16 melanoma cells following oxidative stress. (A) B16 cells were challenged with 0.5 mM H₂O₂ for 20 minutes and allowed to recover in complete medium for up to 8 hours. For TPA treatment, the agent was kept in the medium at a final concentration of 40 nM throughout the experiment, and cells were incubated for times ranging from 0.5 to 8 hours, as indicated on top of each lane. The levels of Mitf mRNA were analyzed by Northern blot. GAPDH mRNA was also analyzed for normalization. C, control untreated cells. Note that the maximal downregulation of Mitf mRNA is similar for TPA and H₂O₂ treatments. Similar trends were obtained in two independent experiments. (B) Quantification of Mitf mRNA variations. Blots were quantified as described in Fig. 7. The results shown correspond to % expression with respect to control cells, and are the mean±range for two determinations. (C) B16 cells were stimulated with the superpotent MSH analogue [Nle⁴, D-Phe⁷]-MSH (Sigma, St Louis, MO), at a final concentration of 100 nM, or with the adenylate cyclase stimulator forskolin (10 µM). Cells were harvested 2 hours after addition of the agents, and Mitf expression was analyzed by Northern blot. C, control; M, MSH-treated cells; F, forskolin-treated cells. (D) Semiquantitative RT-PCR analysis of Mitf mRNA levels in H₂O₂-challenged B16 cells. Cells were treated with H₂O₂, as in A, and allowed to recover for 1, 2, 4 and 8 hours, as indicated. Total RNA was extracted, and cDNA was prepared. Equivalent amounts of cDNA were amplified with primers specific for Mitf and Gapdh, as a control for comparable loading of target cDNA. The reaction mixtures were analyzed by agarose gel electrophoresis. C, control; the lane on the right shows markers of the indicated size.