Figure 5.

Effects of USP7 or USP11 knockdown in INK4a-deficient cells. FDFs or the Leiden strain of INK4a-deficient human fibroblasts (Brookes et al, 2002) were infected with lentiviruses encoding two independent USP7 or USP11 shRNAs (sh1 and sh2) or a control shRNA (Ctrl). The effects on INK4a and the respective target genes were assessed by qRT–PCR (A, B) and immunoblotting (C, D) using an antibody that detects the mutant form of p16^INK4a present in Leiden cells. (E) Phase contrast images showing that FDFs transduced with either USP7 or USP11 shRNAs display a senescence-like phenotype with single prominent nucleoli (arrowheads). Whereas Leiden cells transduced with USP7 shRNAs also display this phenotype, Leiden cells expressing USP11 shRNAs continued to proliferate and resembled cells expressing the control shRNA. (F) Semi-quantitative assessment of the senescence phenotype in FDFs and Leiden cells based on the percentage of cells with a single nucleolus.