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Figure 4. Analysis of TET2 and GLUT1 in the response of A375 cells to ASC. (A) Western blot of GLUT1 protein in A375-P and A375-R; images are representative of three independent western blots; (B) MTT assay of A375-R treated with 0.5 mM ASC in the presence of increasing concentrations of glucose; (C) Western blot of TET2 and GLUT1 in A375-P and A375-R silenced for TET2; images are representative of three independent western blots; (D) Representative images and fluorescence quantification of ROS levels measured by DCFDA in A375-R silenced for TET2 and treated with/without 0.5 mM ASC; Scale bar = 50 µm; (E) Representative images and fluorescence quantification of mitochondrial polarization measured by JC-1 in A375-R silenced for TET2 and treated with/without 0.5 mM ASC; Scale bar = 50 µm. Where indicated, CTRL refers to PBS used as the vehicle control for ASC. Statistical analysis in all bar graphs was performed by one-way ANOVA; (F) Spearman’s correlation analysis performed on melanoma samples from SKCM-TCGA dataset. ^*P < 0.05; ^**P < 0.01; ^***P < 0.001. All experiments were performed in n = 3 independent biological replicates. TET: Ten-Eleven Translocation; ASC: L-ascorbic acid; MTT: 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide; ROS: reactive oxygen species; DCFDA: 2′,7′-dichlorofluorescein diacetate; PBS: phosphate-buffered saline; ANOVA: analysis of variance; SKCM: Skin Cutaneous Melanoma; TCGA: The Cancer Genome Atlas.