Fig. 7. Northern blot analysis of mRNA levels for five melanocyte differentiation markers in H₂O₂-challenged B16 melanoma cells. (A) Representative blots, performed with 10 µg of total RNA from control cells (C), treated exactly as the 8 hours time point or cells treated with H₂O₂ (0.5 mM, 20 minutes) and allowed to recover for 8 or 16 hours. The blots were probed for Tyr, Dct, Tyrp1, silver, Mc1r and ODC mRNA, as indicated, as well as for GAPDH (shown below each lane). Blots were performed in triplicate for Tyr, Dct, Tyrp1 and ODC, and in duplicate for the silver locus product and for Mc1r. (B) Quantification of mRNA variations. Northern blots were quantified by phosphorimaging, in a BioRad GS-525 Molecular Imager. The results, corrected for loading by comparison to the GAPDH signal, are shown as % expression with respect to the control untreated cells, and are the mean±s.d. for the mRNA species analyzed in triplicate (Tyr, Dct, Tyrp1 and ODC). For these species, the statistical significance of the variations was analyzed by calculating two-tailed P values by means of an unpaired Student’s t-test, using the Prism software. , P<0.01. For the species analyzed in duplicate (silver and Mc1r), the values given are the mean±range for the two determinations, and no statistical analysis was performed.