Fig. 3

Vitamin C enhances the efficacy of 5-AZA in inducing active demethylation and generation of 5hmc by induction of TET expression in HCC. a 5hmC nuclear staining of HCC cell lines, HLE and Huh7, treated with vitamin C, 5-AZA, and 5-AZA + vitamin C. 5hmC-positive cells are seen in red, Hoechst is seen in blue. White line represents the scale bar (20 μm). Graphs show the calculation of the percentage of 5hmC-positive cells as an indicator of active demethylation in HLE and Huh7. b Quantitative changes in the mRNA expression of TET2 and TET3 in HLE and Huh7 cells with the various treatments. Data was normalized using GAPDH expression as a reference control. c Western blot results show changes of TET2 and TET3 protein expression in HLE and Huh7 cells with the various treatments. Data was normalized using GAPDH expression as a reference control. All the data are the average of the experiments (N = 3). Statistical significance was tested using one-way ANOVA (non-parametric) Tukey’s test. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.01. Error bars represent the standard deviation