Figure 3.

USP20 is upregulated following DNA damage or replication stress and regulates cell-cycle checkpoint. (A and B) Cells treated as indicated were lysed and cell lysates were then blotted with the indicated antibodies. (C) Cells stably expressing control shRNA or USP20 shRNAs were left untreated or treated with UV (30 J/m²) and harvested at the indicated times. Cells were lysed and cell lysates were then blotted with the indicated antibodies. (D) A549 cells stably expressing control shRNA or USP20 shRNAs were left untreated or treated with UV (30 J/m²). One hour later, cells were lysed and cell lysates were then blotted with the indicated antibodies. (E) A549 cells stably expressing control shRNA or USP20 shRNAs were transfected with the indicated constructs. After 48 h, cells were left untreated or treated with UV (30 J/m²). One hour later, cells were lysed and cell lysates were then blotted with the indicated antibodies. (F) Cells stably expressing Ctrl, USP20shRNA, Claspin shRNA or USP20shRNA together with Claspin shRNA were treated with UV (30 J/m²). One hour later, cells were lysed and cell lysates were then blotted with the indicated antibodies. (G) Cells from (D) were left untreated or treated with HU (10 mM) or UV (30 J/m²). After 2 h, cells were incubated with 20 μM BrdU; 30 min later, cells were fixed and stained with anti-BrdU antibody. (H) Cells stably expressing USP20 shRNA were transfected with shRNA resistant HA-USP20WT or its CA mutant. Cells were left untreated or treated with UV (30 J/m²). After 2 h, cells were incubated with 20 μM BrdU; 30 min later, cells were fixed and stained with anti-BrdU antibody. (I) Cells stably expressing Ctrl, USP20shRNA, Claspin shRNA or USP20shRNA together with Claspin shRNA were left untreated or treated with UV (30 J/m²). After 2 h, cells were incubated with 20 μM BrdU; 30 min later, cells were fixed and stained with anti-BrdU antibody. (G–I) Error bars represent the SEM of three independent experiments. **P < 0.01.