Figure 2.

EZH2 is a substrate of FBW7 in pancreatic cancer cells. A, Western blotting analysis of WCLs of PANC-1 cells transfected with the indicated constructs. Cells were treated with or without 20 μm MG132 for 8 h before harvest. Data are representative of multiple experiments (n = 3). IB, immunoblot. B, PANC-1 and MIA PaCa-2 cells were transfected with control or two independent FBW7-specific shRNAs. 48 h after transfection, cells were harvested for Western blotting analysis. Data are representative of multiple experiments (n = 3). C, PANC-1 cells were transfected with control or shRNAs as indicated. 48 h after transfection, cells were harvested for RT-qPCR analysis of EZH2 and FBW7 mRNAs. Data are mean ± S.D. from experiments with three replicates. *, p < 0.05 compared with the shControl group; n.s., not significant. D and E, PANC-1 cells were transfected with control and FBW7-specific shRNAs. After 48 h, cells were treated with 50 μg/μl cycloheximide (CHX). At different time points, cells were harvested for Western blotting analysis. At each time point, the intensity of EZH2 was normalized to the intensity of β-tubulin (loading control) first and then to the value at the 0-h time point. F, PANC-1 cells were transfected with the indicated plasmids for 24 h, followed by Western blotting analysis. Data are representative of multiple experiments (n = 3). G, PANC-1 cells were transfected with the indicated plasmids for 24 h, followed by Western blotting analysis. Data are representative of multiple experiments (n = 3). H, PANC-1 cells were transfected with the indicated plasmids for 48 h, followed by treatment with 20 μm MG132 for 8 h. Immunoprecipitated EZH2 proteins were analyzed by WB for ubiquitination. I, PANC-1 cells were transfected with the indicated plasmids for 16 h, followed by treatment with 20 μm MG132 for 8 h. Immunoprecipitated FLAG-EZH2 proteins were analyzed by WB for ubiquitination.