Figure 5.

YC-1 enhances proteasome-dependent EZH2 degradation and ubiquitination in breast cancer cells. (A) Effect of YC-1 on EZH2 mRNA abundance in MDA-MB-468 cells. Cells were treated with 3 μM YC-1 for 3 or 6 h. Cells were collected for the detection of EZH2 mRNA expression with Q-RT-PCR. (B and C) Cells were pretreated with cycloheximide (30 μM) for 1 h and followed by induction with vehicle (DMSO) or YC-1 (3 μM) for the indicated times (MDA-MB-468, B) or 10 μM YC-1 for 16 h (SKBR3, C). Cells were harvested for detection of protein level by Western blotting. (D and E) MDA-MB-468 cells were incubated with 3 μM YC-1 for 6 h in the presence of vehicle, 3 μM MG-132, or 30 μM NH₄Cl. Cells were harvested for detection of protein level (D) or assessment of viability (E). The EZH2 expression levels were quantified and are shown under the blot. Results are expressed as mean ± SEM of three independent experiments. *P < 0.05; **P < 0.01, compared with control. (F) After treatment with 3 μM YC-1 for the indicated times, MDA-MB-468 cells were lysed and subjected to the EZH2 ubiquitination assay. (G) After treatment with 10 μM YC-1 for 16 h, SKBR3 cells were collected for the EZH2 ubiquitination assay. Ub, ubiquitin.