Figure 1: USP11 interacts with and stabilizes PML.

(a) Schematic presentation of the screening procedure to identify DUBs that regulate PML. Representative images of cells transduced with lentivirus containing control or USP11 shRNA are shown on the bottom. Scale bar, 20 μm. (b) USP11 shRNAs reduce PML protein but not mRNA. HeLa cells transduced with lentivirus carrying USP11 shRNA or control shRNA were analysed by western blot with indicated antibodies (top) or by reverse transcriptase–quantitative PCR (bottom). Data shown are mean±s.d. of three independent experiments. (c) HeLa cells transduced with indicated lentiviruses were treated with 1 μM MG132 for 18 h and analysed by western blot. (d,f) The effects of USP11 knockdown (d) or overexpression (f) on PML half-life. HeLa cells stably expressing indicated shRNAs (d), or U87 cells transfected with indicated constructs (f) were treated with 100 μg ml⁻¹ cycloheximide for indicated time periods and were analysed by western blot. The levels of PML relative to that seen at 0 h were quantified and plotted on the left panels and the PML half-life in each cell is indicated on the bottom. (e) Western blot analysis of PML level in HeLa cells transfected with indicated constructs. (g) USP11 interacts with PML-I and PML-IV. Coimmunoprecipitation analysis of 293T cells transfected with indicated constructs. (h) Interaction of endogenous PML with endogenous USP11 in T98G GBM cells and H4 glioma cells was analysed by coimmunoprecipitation. Lysate of PML-null MEFs was used as a control. (i) USP11 and PML interact in vitro. Baculovirally purified USP11 bound on Myc-agarose beads was used to pull down baculovirally purified PML-I. Bound proteins were analysed by western blot with indicated antibodies. (j) Mapping of the USP11 domain involved in PML interaction. The domain organization of USP11 is shown on the top panel. Interaction of PML-I with indicated USP11 mutants in transfected 293T cells was analysed by coimmunoprecipitation (bottom panels).