Fig 1: USP7-mediated IKZF1 stabilization is potentiated by DNA damage. (A-B) IKZF1-KD or USP7-KD NCI-H929 cells transfected with the control vector, HA-IKZF1 (IKZF1/WT) were subjected to IR (5 Gy) and examined for foci of RPA2 (A) and RAD51(B) by IF at the indicated time points. Scale bar: 20 µm. Quantitation of RPA2- or RAD51-positive cells (foci >15) is shown. Data are mean ± s.d. p-values were analyzed by one-way analysis of variance (ANOVA) and two-sided Student's t-test. *p < 0.05, ns: no significant. (C-E) USP7-KD NCI-H929 cells transfected with control vector, HA-IKZF1 (IKZF1/WT) were subjected to IR (5 Gy) and examined for the γH2AX foci at the indicated time points. Scale bar: 20 µm. Quantitation of γH2AX positive cells (foci >10) is shown. (F-G) HEK293T cells stably expressing Flag-IKZF1 were co-transfected with HA-Ub/WT followed by CPT (100 nM) for 1 h (F) or IR (5 Gy) (G) treatment. Cellular extracts were immunoprecipitated with anti-Flag antibody and then immunoblotted with the indicated antibodies. Data are mean ± s.d. p-values were analyzed by one-way analysis of variance (ANOVA). *p < 0.05, **p < 0.01, ***p< 0.001, ****p< 0.0001, ns: no significant.
Fig 2: P5091 sensitizes multiple myeloma cells to Lenalidomide in vitro and in vivo. (A-E) NCI-H929 (A), MM1.S (B), CD138+ MM patient cells (C, D), and normal BM mononuclear cells (E) were cultured in the control medium or in the presence of P5091 and/or olaparib for 48 h. The cell viability was determined by the CCK-8 kit. Data were analyzed online (https://synergyfinder.fimm.fi). (F-J) RPMI-8226 cells were subcutaneously injected into the flank of NOD-SCID mice. Mice were treated with the vehicle, P5091 (10 mg/kg i.v.) and/or olaparib (50 mg/kg i.p.) (F). Mice tumor volume (G), tumor images (H), tumor weight (I), and body weight (J) were then assessed. (K) Lenalidomide or USP7i sensitizes multiple myeloma cells to PARPi in vitro and in vivo. USP7i: USP7 inhibitor; PARPi: PARP inhibitor. Data are mean ± s.d. p-values were analyzed by two-way analysis of variance (ANOVA). *p < 0.05, ***p < 0.001, ns: no significant.
Fig 3: IKZF1 is physically associated with USP7. (A) Immunopurification and mass spectrometric analysis of IKZF1-interacting proteins. The asterisks denote the nonspecific IgG heavy-chain signal. (B, C) Co-IP analysis of the association between IKZF1 and USP7. Whole-cell lysates from HEK293T cells stably expressing Myc-IKZF1 an Flag-USP7, as well as RPMI-8226 and MM1.S cells were immunoprecipitated and immunoblotted with antibodies against the indicated proteins. (D) Detection of 3×Flag-IKZF1 bound to GST or GST-His-USP7 in a GST pull-down assay. (E) Confocal microscopic analysis of IKZF1 and USP7 subcellular localization. RPMI-8226 cells were fixed and immunostained with antibodies against the indicated proteins. Representative images from biological triplicate experiments are shown. Scale bars: 20 µm. (F) HEK293T cells stably expressing HA-IKZF1 were transfected with GFP-USP7 WT, GFP-USP7 ?MATH, or GFP-USP7 ?UBL, or GFP-USP7 CD, respectively. Cellular extracts were immunoprecipitated with anti-GFP antibody followed by immunoblotting (IB) with indicated antibodies. Domain names are: MATH: TRAF homology domain, CD: Catalytic domain, UBL: Ubiquitin-like domains. (G) HEK293T cells stably expressing Flag-USP7 were co-transfected with the indicated truncates of IKZF1, respectively. Cellular extracts were immunoprecipitated with anti-HA antibody followed by IB with indicated antibodies. Zinc fingers are depicted in blue boxes.
Fig 4: USP7 deubiquitinates IKZF1. (A) RPMI-8226, MM1.S, and NCI-H929 cells were cultured in the absence or presence of the indicated doses of P5091 for 7 hours. Cellular extracts were collected for western blotting with indicated antibodies. (B) RPMI-8226 and MM1.S cells were treated with P5091 (15 µM) for 6 h and further incubated with or without MG132 (5 µM) for another 3 h; the indicated proteins were examined by western blotting. (C) RPMI-8226 cells were transfected with the control shRNA or USP7 shRNAs, (D) or overexpressed with Flag-USP7. Cellular extracts were collected for western blotting with indicated antibodies. (E-F) HEK293T cells stably transfected with HA-IKZF1 were co-transfected with an increasing dose of Flag-USP7 (E) or its inactive C223S mutant (F). Cellular extracts were collected for western blotting with the indicated antibodies. (G) HEK293T cells stably expressing Flag-USP7 were co-transfected with the indicated IKZF1 truncates, respectively. Cellular extracts were collected for western blotting with the indicated antibodies. (H) RPMI-8226 cells stably expressing control shRNA or USP7 shRNAs were treated with DMSO or MG132 (5 µM) for 3 h. Cellular extracts were immunoprecipitated with anti-IKZF1 followed by IB with indicated antibodies. (I) HEK293T cells stably expressing Myc-IKZF1 were co-transfected with HA-Ub/WT and Flag-USP7/WT or Flag-USP7/C223S as indicated. Cellular extracts were immunoprecipitated with anti-Myc antibody followed by IB with indicated antibodies. The asterisks denote the nonspecific IgG heavy-chain signal. (J) HEK293T cells stably expressing Myc-IKZF1 were co-transfected with His-Ub WT and Flag-USP7 WT or Flag-HA-USP47 as indicated. Cell lysates were subjected to western blotting with the indicated antibodies. (K) HEK293T cells stably expressing Myc-IKZF1 were co-transfected with HA-Ub/WT and Flag-USP7/WT and treated with P22077 (10 µM) for 6 h before being harvested. Then, cellular extracts were immunoprecipitated with anti-Myc antibody followed by western blotting with the indicated antibodies. (L) In vitro deubiquitination assays were performed with His-Ub-conjugated IKZF1 purified from HEK293T cells and recombinant GST-USP7/WT (0.5 µg) at 37 °C for 2 h, followed by western blotting with the indicated antibodies. Data are mean ± s.d. p-values were analyzed by one-way analysis of variance (ANOVA) (A, C) and two-sided Student's t-test (D). ns: no significant.
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