Figure 1.

Exogenous oxidative stress acutely upregulates p16 in human skin cells, and melanocytes are more susceptible than other cell types to oxidative stress and damage. (a) Melanocytes were untreated (0 h) or UV-irradiated (480 J/m², lanes 2-5), and cell lysates were prepared over a 24-h period and blotted with antibodies against p16 and Actin (upper panel). Melanocytes were treated with H₂O₂ at the concentrations indicated, in the absence (lanes 1-3) or presence (lanes 4-6) of 5 mM NAC, and 5 h later cell lysates were blotted for p16 and Actin (lower panel). (b) Melanocytes were untreated (0 h) or UV-irradiated (480 J/m²) in the absence or presence of 5 mM NAC, and 5 h later ROS levels were measured by DCFDA assay (upper panel, values normalized to mean of control conditions which were set at 1) and cell lysates were blotted for p16 and Actin (lower panel). Error bars indicate SEM from three independent experiments. *P<.001 (one-sample t test), **P<.001 (two-sample t test). (c) Keratinocytes (KC), fibroblasts (FB), and melanocytes (MC) isolated from each of six donors were untreated or treated with 0.05 mM H₂O₂ for 1.5, 3, or 5 h. RNA was isolated and expression of p16 and GAPDH was quantitated by qRT-PCR, with p16 levels normalized to GAPDH at each time point (and then normalized to control conditions which were set at 1). Error bars indicate SEM from six independent determinations. *P<.05 (one-sample t tests). (d) Melanocytes (MC), keratinocytes (KC), and fibroblasts (FB) isolated from each of seven donors were untreated or treated with 0.05 mM H₂O₂ for 5 h, and ROS levels were measured by DCFDA assay (left panel). Error bars indicate SEM from seven independent determinations. *P<.001 (repeated measures ANOVA analysis, P values adjusted for multiple comparisons). Cells isolated from each of three donors were untreated or treated with 0.5 mM H₂O₂ for 48 h, then fixed and immobilized for 8-OG staining. Error bars indicate SEM of percent 8-OG positive cells assessed under each condition from three independent determinations. *P=.06, **P<.001 (adjusted for multiple comparisons).