Figure 4. Catalytically inactive USP7 stimulates p53 function.

(A) U2OS cells were transfected with an empty vector or vector expressing the myc-tagged C223S mutant of USP7 and treated with etoposide for 0, 1, 2 or 4 hours as indicated. Equal amounts of cell lysates were analyzed for protein expression by western blotting using the indicated antibodies where actin is the loading control. (B) H1299 cells were either transfected with an empty plasmid (Vector) or transfected with a p53-expressing plasmid alone or in combination with constructs expressing myc-tagged USP7 and USP7 mutants as indicated. 24 hours post-transfection, cells were lysed and protein levels were measured by Western blotting using the antibodies indicated. The higher p53 band in lane 4 corresponds to monoubiquitylated p53 which becomes apparent due to the dominant-negative effects of C223S [24]. (C) H1299 cells were transfected with plasmids expressing p53 and myc-tagged USP7-CTD and a luciferase reporter construct in the combinations indicated. 48 hours post-transfection, cells were lysed and luciferase activity was quantified. The results are shown relative to the p53 plus reporter sample for three independent experiments.