Fig. 4.

Guanine deaminase (GDA) metabolic products including uric acid enhanced keratinocyte senescence and reactive oxygen species (ROS) generation. (a) Western blot analysis showing the relative ratios of xanthine and uric acid levels in primary cultures of normal human skin keratinocytes with or without GDA overexpression. (b-c) Representative immunofluorescent staining using (b) anti-uric acid antibody, (c) western blot analysis showing the relative ratios of p16 and p21 and immunofluorescent staining using anti-GDA, anti-p16, or anti-p21 antibody in keratinocytes (KCs) overexpressing wild-type GDA or deletion mutants (Δ75-84 or Δ75-84 and 350-402) of GDA, in which GDA protein levels were identified by western blot analysis. (d-e) Assay for uric acid concentration (d) and western blot analysis showing the relative ratios of p16 and p21 protein levels and dihydroethidium (DHE) staining in KCs with or without xanthine and allopurinol (e). β-Actin was used as an internal control in western blot analysis. Nuclei were counter-stained with Hoechst 33258 (bar 0.05 mm) and staining intensities were measured using Wright Cell Imaging Facility (WCIF) ImageJ software. Data represent mean ± standard deviation of 3-4 independent experiments. *p < 0.05 vs. control KCs, #p < 0.05 vs. wild-type GDA-overexpressing KCs or KCs treated with xanthine.