fig5

Figure 5. Analysis of ASC response mechanisms in M14 melanoma cells. MTT assay of M14-P and M14-R treated with (A) 0.5 and 1 mM ASC or (B) 0.5 and 1 mM p-ASC; (C) Representative images and fluorescence quantification of ROS levels measured by DCFDA in M14-P and M14-R treated with/without 0.5 mM ASC or p-ASC; Scale bar = 50 µm; (D) Fluorometric analysis of GSH/GSSG ratio in M14-P and M14-R treated with/without 0.5 mM ASC; (E) Western blot of SOD2 and CAT proteins in M14-P and M14-R; images are representative of three independent western blots; (F) Direct cell counting of M14-R cells silenced for TET2 with/without 0.5 mM ASC; (G) Representative images and fluorescence quantification of ROS levels measured by DCFDA in M14-R silenced for TET2 and treated with/without 0.5 mM ASC; Scale bar = 50 µm; (H) Western blot of GLUT1 proteins in M14-P and M14-R; images are representative of three independent western blots; (I) MTT assay of M14-R treated with 0.5 mM ASC in the presence of increasing concentrations of glucose; (J) Western blot of TET2 and GLUT1 in M14-P and M14-R silenced for TET2; images are representative of three independent western blots. Where indicated, CTRL refers to PBS used as the vehicle control for ASC and/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; MTT: 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide; p-ASC: L-ascorbic acid 2-phosphate; ROS: reactive oxygen species; DCFDA: 2′,7′-dichlorofluorescein diacetate; SOD2: superoxide dismutase 2; CAT: catalase; TET: Ten-Eleven Translocation; PBS: phosphate-buffered saline; ANOVA: analysis of variance.