Figure 7.

YC-1 activates c-Cbl to mediate EZH2 down-regulation in breast cancer cells. (A) c-Cbl-knockdown cells were treated with YC-1 (MDA-MB-468: 3 μM for 4 h; SKBR3: 10 μM for 10 h). Cells were harvested for detection of protein level by Western blotting. The EZH2 expression levels were quantified and are shown under the blots. (B) MDA-MB-468 cells were treated with 3 μM YC-1 for the indicated times. Cells were collected and c-Cbl phosphorylation was detected. (C) MDA-MB-468 cells were pretreated with MG-132 (3 μM) or NH₄Cl (30 μM) for 1 h followed by 3 μM YC-1 induction for 6 h and c-Cbl was detected by Western blotting. (D) MDA-MB-468 cells were pretreated with MG-132 and then induced by 3 μM YC-1 for the indicated times. Cells were harvested and lysed for the immunoprecipitation assay. The association among c-Cbl, ERK and EZH2 was determined by Western blotting and probing with anti-EZH2, anti-ERK or anti-c-Cbl. Membranes were stripped and reprobed with anti-c-Cbl or anti-EZH2 to check the input. (E) Cells were pre-incubated with or without PD98059 (PD, 10 μM) in the presence of MG-132 for 1 h before treatment with 3 μM YC-1 for 2 h. Cells were then collected and the c-Cbl-EZH2 complexes were detected in the lysate protein.