Fig. 4

Hinokitiol increases TET1 expression via enhancement of 5hmC level in HCT-116 cells. HCT-116 cells were treated with 5 and 10 μM of hinokitiol for 72 h and nuclear protein isolated from the cells was used to detect TET1 expression using western blot analysis (a). The expression level of each protein was quantified with the Image Studio Lite program, using Lamin B as a loading control. The histogram shows the quantification expressed as ratio of the intensity of target gene/Lamin B. Experiments were done twice and representative blots were shown. Contents of 5hmC measured from cells treated with 10 μM of hinokitiol for 72 h using ELISA-based methylflash hydroxymethylated DNA quantification kit (b). The levels of 5mC and 5hmC were confirmed using flow cytometry analysis (c). All data are representative of three independent experiments performed in duplicate. The results were representative of three independent experiments. Data are the means ± SE of results from at least three independent experiments. * indicates a significant difference at the level of < 0.05