Figure 6

ROS regulates TET mRNA expression and TET protein activities.

(a) DHE staining to evaluate ROS levels in MGC803 cells treated with indicated concentrations of H₂O₂. Scale bar = 100 μm.(b) Relative fluorescent density of cells stained by DHE in (a). Statistical comparisons between groups were conducted by unpaired Student’s t-test. P-values were shown in the figure. *indicates p < 0.05; ***indicates p < 0.01. (c) qRT-PCR was performed to test relative TET mRNA expression of MGC-803 cells treated with indicated concentration of H₂O₂. ACTB was chosen as a loading control. Data are expressed as mean ± SEM. Statistical comparisons between groups were conducted by one-way ANOVA followed by a Newman-Keuls comparison test. P-values were shown in the figure. *indicates p < 0.05; **indicates p < 0.01. The value of p < 0.05 was considered to be significant.(d) HEK 293FT cells were transfected with GFP-tagged TET1, Flag-tagged TET2 and Flag-tagged TET3 expression plasmid respectively and cultured for 24 h, then the cells were treated with indicated final concentrations of H₂O₂ for 5 min. Cells were lysed in reduced and non-reduced conditions followed by western blot analysis. Molecular weights were determined by a simultaneously run protein ladder. (e) HEK 293FT cells ectopic expressing GFP-tagged TET1-ΔCXXC (TET1 mutation with CXXC domain deletion) and HA-tagged TET1-CD (TET1 mutation with only CD domain) were treated with H₂O₂ at indicated concentrations for 10 min respectively. Lysates were analyzed by western blot with GFP and HA antibody under reducing or non-reducing conditions. (f) The oxidative stress induced by H₂O₂ was indicated by Prdx-SO₃ level as a positive control.