Figure 4.

Knockdown of USP7 or USP11 increases expression of INK4a. (A, B) Knockdown of USP7 (A) or USP11 (B) in FDF cells using two different shRNAs (sh1 and sh2) leads to up-regulation of p16^INK4a protein (left panels) and INK4a RNA (right panels) with no equivalent effect on ARF RNA. (C) Chromatin immunoprecipitation showing that knockdown of USP7 or USP11 results in decreased binding of MEL18 to the INK4a locus. The map of the INK4a-ARF locus and PS numbers show the location of previously described primer sets used to interrogate the precipitated DNA by qPCR (Bracken et al, 2007). IgG shows the maximum background signal obtained using an irrelevant rabbit antibody. (D) FDFs transduced with independent shRNAs against USP7 and USP11 show impaired proliferation relative to cells expressing control shRNA, as judged by crystal violet staining. Each time point represents an average of six replicates and the A_{595 nm} values were normalised to the absorbance at day 0 (first day after plating). (E) SA-β-Gal staining of FDFs expressing control shRNA (Ctrl) or two independent shRNAs against USP7 or USP11.