Exogenously expression of DNMT1 or EZH2 feedback reversed the effect of β-elemene on phosphorylation of Stat3 and cell growth inhibition. (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 levels of DNMT1 and EZH2, and p-Stat3, Stat3 proteins using corresponding antibodies. (C,D) 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 48 h. Afterwards, cell proliferation was examined by MTT assays as described in the Materials and Methods section. Insert blots represented protein expressions of DNMT1 and EZH2. (E,F) C666-1 cells were transfected with control or DNMT1 or EZH2 siRNAs for 30 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 levels of DNMT1 and EZH2, and p-Stat3, Stat3 protein 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).