Figure 4.

Exogenously expression of DNMT1 overcame the effect of β-elemene on EZH2 protein expression and promoter activity. (A,B) C666-1 and HNE2 cells were transfected with control or DNMT1 or EZH2  expression vectors for 24 h prior to exposure of the cells to β-elemene (20 μg/mL) for an additional 24 h. Afterwards, Western blot analysis were used measure the protein levels of DNMT1 and EZH2 using corresponding antibodies. (C) C666-1 and HNE2 cells were transfected with control or DNMT1 expression vector, and with a wild type human EZH2 promoter reporter construct ligated to luciferase reporter gene and internal control secreted alkaline phosphatase for 24 h, followed by treating with β-elemene (20 μg/mL) for an additional 24 h. Afterwards, the promoter activities were determined using the Secrete-Pair Dual Luminescence Assay Kit as described in the Materials and Methods section. (D) C666-1 cells were transfected with control or DNMT1 siRNA for 24 h prior to exposure of the cells to β-elemene (20 μg/mL) for an additional 24 h. Afterwards, Western blot analysis were used for determining the protein levels of DNMT1 and EZH2 using corresponding antibodies. The figures are representative cropped gels/blots that have been run under the same experimental conditions. Values in bar graphs were given as the mean ± SD from three independent experiments performed in triplicate. *Indicates significant difference as compared to the untreated control group (P < 0.05). **Indicates significant difference from the β-elemene treated alone (P < 0.05).