Extended Data Figure 7 : USP7 knockdown or inhibitors affect USP7 substrates in cell lines.

From: Molecular basis of USP7 inhibition by selective small-molecule inhibitors

Extended Data Figure 7

a, HCT116 cells were transfected with non-targeting control (NT1) or three different USP7-targeting siRNAs for 72 h, harvested and probed with indicated antibodies. A representative experiment from two biological replicates is shown. b, U2OS cells were transfected with a pool of four independent siRNAs targeting USP7 or a non-targeting control siRNA and processed as in a. A representative experiment from two biological replicates is shown. c, HCT116 cells were treated with 10 μM FT671 for 24 h and RNA was extracted for qPCR measurements with primer sets against indicated transcripts (see Methods). Data were analysed with the ΔCt method. Experiments were performed in triplicate. d, HCT116 cells were treated with FT671 (10 μM) for longer periods, harvested and probed with indicated antibodies. A representative experiment from two biological replicates is shown. e, Experiment as in Fig. 4d, with higher FT671 concentration. p53 levels are upregulated upon FT671 treatment in cells expressing GFP or GFP-tagged wild-type USP7, but not in cells expressing compound resistant mutants. USP7 expression was induced for 7 h by 1 μg ml−1 doxycycline, and cells were treated with 3 μM FT671 for the last 4 h before lysis. f, IMR-32 neuroblastoma cells33 were treated with indicated concentrations of FT671, and effects on p53 and N-Myc levels were tested by western blotting. A representative experiment from two biological replicates is shown. g, h, HCT116 (g) or MM.1S (h) cells were treated for indicated times with FT671, and western blotted for UHRF1 and DNMT1. Vinculin and β-actin serve as loading controls. A representative experiment from two biological replicates is shown. Uncropped images for gels are shown in Supplementary Fig. 1.