RNA-seq Reveals Dysregulation of Novel Melanocyte Genes upon Oxidative Stress: Implications in Vitiligo Pathogenesis

Figure 1

Characterization of melanocytes and H₂O₂-induced oxidative death: (a) PIG1 and primary HEM were incubated with L-DOPA or PBS (control) for 5 h and images were obtained using phase contrast microscopy. Note that the melanocytes are branched and stained black by L-DOPA, confirming the presence of DOPA oxidase (tyrosinase) activity. (b) Indicated cells were cultured, and cell lysates were probed with MITF and TYRP-1 antibodies using western blotting. Equal loading was confirmed using β-actin antibodies. (c) PIG1 melanocytes were either left untreated or incubated with indicated concentration of H₂O₂ for 2 h at 37°C. The oxidative stress was detected by staining cells with CellROX Orange dye for 30 min, and images were obtained by the Evos fluorescent microscope. (d) To observe the morphological changes, phase contrast microscopic images were obtained 3 h after exposure to H₂O₂. (e) PIG1 melanocytes were cultured with indicated concentration of H₂O₂ for 6 h, 24 h, and 48 h. Cell viability was checked by trypan blue dye exclusion assay. Mean (three independent observations) values of viability percentages were plotted at different time intervals. Greater reduction in cell viability was found with increasing concentration of H₂O₂. Grey box includes the time points chosen for RNA-seq experiments. (f) Flow chart showing the different time points and concentrations of H₂O₂ used in RNA-seq experiments.

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