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Figure 3. Analysis of ASC-induced ROS production and antioxidant response in A375 cells. (A) MTT assay of A375-P and A375-R treated with 0.5 and 1 mM p-ASC; (B) Representative images and fluorescence quantification of 5hmC levels in A375-P and A375-R treated with/without 0.5 mM ASC or p-ASC. Scale bar = 50 µm; (C) Representative images and fluorescence quantification of ROS levels measured by DCFDA in A375-P and A375-R treated with/without 0.5 mM ASC or p-ASC. Scale bar = 50 µm; (D) Fluorometric analysis of GSH/GSSG ratio in A375-P and A375-R treated with/without 0.5 mM ASC; (E) Western blot of SOD2 and CAT proteins in A375-P and A375-R; images are representative of three independent western blots; (F) NAC or (G) Trolox treatment in the presence of 0.5 or 1 mM ASC and analysis of cell viability by MTT assay in A375-R cells. Where indicated, CTRL refers to PBS used as the vehicle control for ASC or p-ASC. Statistical analysis in all bar graphs was performed by one-way ANOVA. ^*P < 0.05; ^**P < 0.01; ^***P < 0.001. All experiments were performed in n = 3 independent biological replicates. ASC: L-ascorbic acid; ROS: reactive oxygen species; MTT: 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide; p-ASC: L-ascorbic acid 2-phosphate; 5hmC: 5-hydroxymethylcytosine; DCFDA: 2′,7′-dichlorofluorescein diacetate; SOD2: superoxide dismutase 2; CAT: catalase; NAC: N-acetylcysteine; PBS: phosphate-buffered saline; ANOVA: analysis of variance; DAPI: 4′,6-diamidino-2-phenylindole.