FIG 5.

Effect of USP7 inhibition on PRC1 levels and stability. (A) Western blot analysis of the expression of USP7, SCML2, RING1B, BMI1, CBX2, CBX8, and the PRC2 component EZH2 after treatment with the USP7 inhibitor P22077 for 4 or 8 h at the indicated concentrations. (B) HCT116 cells were treated with DMSO or 50 μM P22077 for 6 h and then treated with cycloheximide and either DMSO or P22077. Cells were collected every 2 h, and whole-cell extracts were analyzed by Western blotting for the expression of USP7, SCML2, RING1B, BMI1, CBX2, and the PRC2 component EZH2. p53 and histone H2A are shown as controls. (C) Changes in the levels of RING1B were quantified and normalized by the amount of USP7. The means of data from 2 independent experiments are shown. (D) Analysis by Western blotting of histone H2A and ubiquitinated histone H2A under the same conditions as those described above for panel A.