YC-1 down-regulates EZH2 expression in breast cancer cells. (A and B) MDA-MB-468 (A and B) and SKBR3 (A) cells were treated with the indicated concentrations of YC-1 (MDA-MB-468: 24 h; SKBR3: 30 h; A) or 3 μM YC-1 for the indicated times (B). Cells were harvested and protein content analysed by Western blotting. The levels of EZH2 expression were quantified and are shown under the blots. MDA-MB-468 (C) and SKBR3 (D) cells were transfected with EZH2 shRNA for 0, 1, 2, 3 and 4 days, and collected for determination of cell viability (left) and protein level (right). (E and F) MDA-MB-468 cells were transfected with shRNA for 72 h, and then floating cells were removed by washing. Cells were then incubated with 3 μM YC-1 for 4 h and analysed for viability (E) and protein content (F). Results are expressed as mean ± SEM of three independent experiments. **P < 0.01, compared with control (shLuc). (G) Tumour specimens were isolated and protein was extracted for Western blotting. Results are expressed as mean ± SEM of six mice. **P < 0.01, compared with control.