Journal: The Journal of Clinical Investigation

Article Title: Destabilizing NEK2 overcomes resistance to proteasome inhibition in multiple myeloma

doi: 10.1172/JCI98765

Figure Lengend Snippet: (A) HEK293T cells were transfected with either empty vector (EV) or HA-FLAG(3×)-NEK2. Proteins binding to NEK2 were pulled down by tandem HA and FLAG antibodies and stained with silver prior to mass spectrometry. (B) ARP1 myeloma cells were lysed and NEK2 was immunoprecipitated using NEK2 antibodies. Western blots were probed with NEK2 and USP7 antibodies. FT, LW, and E represent flow through, last wash, and elution of the immunoprecipitation, respectively. (C) ARP1 myeloma cells were transduced with NEK2-HA plasmids. Transduced cells were lysed and NEK2 was immunoprecipitated using HA antibodies. Western blots were probed using NEK2 and USP7 antibodies. (D) H1299 cells were transfected with mock or USP7-FLAG overexpression vector. Endogenous NEK2 was immunoprecipitated and Western blots were analyzed using NEK2 and USP7 antibodies. (E) ARP1 myeloma cells transduced with EV + USP7-shRNA or NEK2-OE + USP7-shRNA were treated with doxycycline (DOX) or vehicle to suppress USP7 expression. After 72 hours, cells were treated with bortezomib (BTZ; 5 nM) for a further 24 hours and cell viability was measured using trypan blue stain. (F) OPM2 cells transduced with NEK2-shRNA were treated with DOX or vehicle to suppress NEK2 expression. ARP1 cells or OPM2 cells with or without silencing of NEK2 were treated with BTZ (2.5, 5, and 10 nM) for a further 24 hours and cell viability was measured using trypan blue stain. Viability experiments were performed in triplicate and a Student’s t test was performed and showed the significance at 10 nM with or without silencing of NEK2. *P < 0.05.

Article Snippet: The USP7-Flag vector was obtained from Addgene (plasmid 16655).

Techniques: Transfection, Plasmid Preparation, Binding Assay, Staining, Mass Spectrometry, Immunoprecipitation, Western Blot, Transduction, Over Expression, shRNA, Expressing

Journal: The Journal of Clinical Investigation

Article Title: Destabilizing NEK2 overcomes resistance to proteasome inhibition in multiple myeloma

doi: 10.1172/JCI98765

Figure Lengend Snippet: (A and B) Knockdown of USP7 decreases NEK2 protein. ARP1 (A) and OCI-MY5 (B) myeloma cells were transfected with EV, NEK2, or NEK2 + USP7-shRNA. After 72-hour induction with doxycycline, cells were lysed. NEK2 and USP7 protein levels were analyzed by Western blot. (C) OCI-MY5, Delta-47, JJN3, OPM2, and ARP1 myeloma cell lines were treated with 16 μM P5091 for 24 hours. Cells were lysed and NEK2 levels analyzed by Western blot. (D) H1299 cells were transfected with mock or USP7-FLAG–overexpressing vectors, lysed, and NEK2 and USP7 levels were determined by Western blot. (E) ARP1 myeloma cells were treated with the proteasome inhibitor MG132 (10 μM) alone for 30 minutes or in combination with P5091 (16 and 25 μM) for an additional 5 hours. Cells were lysed and NEK2 levels were analyzed by Western blot. (F) OPM2 cells were treated with or without P5091 (25 μM for 2 hours) and protein was extracted with lysis buffer supplemented with NEM. Endogenous NEK2 was immunoprecipitated and analyzed by Western blot using NEK2 and ubiquitin antibodies. FT, LW, and E represent flow through, last wash, and elution of the immunoprecipitation, respectively. (G) H1299 cells were transfected with EV and HA-ubiquitin (HA-UB) or FLAG-USP7 and HA-UB. Cells were lysed and endogenous NEK2 was immunoprecipitated (IP) by NEK2 antibodies and ubiquitination levels were analyzed by Western blot. The higher-molecular-weight band is nonspecific IgG. (H) H1299 cells were transfected with NEK2-OE, HA-UB, and FLAG-USP7 or NEK2-OE and HA-UB. Cells were lysed and total NEK2 protein, including both endogenous and exogenous, was immunoprecipitated (IP) by anti-NEK2 antibodies and ubiquitination levels were analyzed using HA antibodies by Western blot.

Article Snippet: The USP7-Flag vector was obtained from Addgene (plasmid 16655).

Techniques: Transfection, shRNA, Western Blot, Lysis, Immunoprecipitation, Molecular Weight

Journal: The Journal of Clinical Investigation

Article Title: Destabilizing NEK2 overcomes resistance to proteasome inhibition in multiple myeloma

doi: 10.1172/JCI98765

Figure Lengend Snippet: (A) Primary myeloma samples from 16 patients (Pts) were lysed and analyzed by Western blot using NEK2, p-p65-S536, total p65 (p65), USP7, and GAPDH antibodies. (B) CD138-positive myeloma cells isolated from 4 primary myeloma patients were mounted on cytospin slides and analyzed by immunofluorescence using NEK2 and p-p65-S536 antibodies. DAPI staining was used to visualize nuclei. Yellow arrowheads indicate myeloma cells coexpressing NEK2 and p-p65-S536. Blue arrowheads show myeloma cells expressing p-p65-S536 with undetectable NEK2 levels. (C) EV and NEK2-OE ARP1 cells were treated with vehicle, BAY11-7082 (0.5 or 1.0 μM), and bortezomib (5 nM) alone or in combination. After 48 hours, cell viability was assessed by trypan blue staining and Dunnett’s method was used to calculate the multiplicity-adjusted P values for each treatment and control group pair. **P = 0.0023; ****P = 0.0001. NS, no significance. Experiment was performed in triplicate. (D) A model for NEK2 deubiquitination and stabilization by interacting with USP7. USP7 prevents E3 ligase APC/C (30) to ubiquitinate NEK2 resulting in its stabilization.

Article Snippet: The USP7-Flag vector was obtained from Addgene (plasmid 16655).

Techniques: Western Blot, Isolation, Immunofluorescence, Staining, Expressing

Journal: The Journal of Clinical Investigation

Article Title: Destabilizing NEK2 overcomes resistance to proteasome inhibition in multiple myeloma

doi: 10.1172/JCI98765

Figure Lengend Snippet: (A) USP7 was knocked down in ARP1 cells transduced with NEK2-OE after 72 hours induction with doxycycline (DOX). Nuclear and cytosolic fractionations were carried out. p65 levels were analyzed between EV and NEK2-OE with or without USP7 shRNA by Western blot. β-Actin and histone H3 (H3) were used as cytosolic and nuclear markers, respectively. (B–D) EV and NEK2-OE ARP1, OCI-MY5, and H1299 cells were lysed. NEK2, p65-S536 phosphorylation, IKK phosphorylation, and IκBα were analyzed by Western blot. (E) H1299 cells transiently transfected with EV or NEK2-OE (WT) or NEK2-K37R mutant (NEK2-Dead) were lysed, and NEK2 and p65-S536 phosphorylation was analyzed by Western blot. (F) ARP1 and OCI-MY5 cells transfected with EV or NEK2-OE were treated with vehicle or MK-2206 2HCl, an AKT inhibitor, for 30 minutes and then cells were lysed. p65-S536 phosphorylation was analyzed by Western blot. (G) NEK2-shRNA ARP1 cells were induced with DOX for 48 hours and then treated with tautomycin, a PP1α inhibitor, for another 24 hours. NEK2, p-p65-S536, p-PP1α, and p-AKT were analyzed by Western blot.

Article Snippet: The USP7-Flag vector was obtained from Addgene (plasmid 16655).

Techniques: Transduction, shRNA, Western Blot, Transfection, Mutagenesis

Journal: The Journal of Clinical Investigation

Article Title: Destabilizing NEK2 overcomes resistance to proteasome inhibition in multiple myeloma

doi: 10.1172/JCI98765

Figure Lengend Snippet: (A) EV and NEK2-OE ARP1 and OCI-MY5 cells were treated with vehicle or BSM-345541, and HSPE mRNA levels were analyzed by qRT-PCR. (B) HPSE mRNA levels were analyzed by qRT-PCR in EV, NEK2-OE, or NEK2-OE + USP7-shRNA ARP1 and OCI-MY5 myeloma cells. **P < 0.01 by Student’s t test. (C) ARP1 and (D) OPM2 cells were treated with P5091 (16 μM overnight), INH1 (25 μM for 24 hours), or NEK2-shRNA DOX (48 hours). Cells were lysed and proteins were analyzed by Western blot with NEK2, p-p65-S536, HPSE, and GAPDH antibodies.

Article Snippet: The USP7-Flag vector was obtained from Addgene (plasmid 16655).

Techniques: Quantitative RT-PCR, shRNA, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: Destabilizing NEK2 overcomes resistance to proteasome inhibition in multiple myeloma

doi: 10.1172/JCI98765

Figure Lengend Snippet: USP7 binds to and stabilizes NEK2 by deubiquitination, allowing it to accumulate in myeloma cells. Accumulated NEK2 binds to and phosphorylates PP1α, resulting in loss its AKT-suppressing activity. Active AKT triggers the canonical NF-κB pathway by phosphorylating IKK, with subsequent phosphorylation and degradation of IκBα. p65 released from the complex with IκBα translocates into the nucleus, where it activates its target genes leading to drug resistance in myeloma. Ub, ubiquitin.

Article Snippet: The USP7-Flag vector was obtained from Addgene (plasmid 16655).

Techniques: Activity Assay

Journal: bioRxiv

Article Title: Identification and validation of E3 ubiquitin ligase XIAP as a novel substrate of deubiquitinase USP7 (HAUSP) - Implication towards oncogenesis

doi: 10.1101/2021.08.12.456108

Figure Lengend Snippet: A, HCT116 (p53 Wt and p53 Null ) lines were treated with the indicated dose of P5091 (0 to 80 µM) for 24 and 48 hrs, and cell viability was checked by MTT assay. Inset graph representing change in mean IC-50 value of both cell lines at different time points. B, Protein levels of USP7, PARP, Cleaved PARP, Caspase3, Cleaved Caspase3, p53, MDM2 and p21 were determined by immunoblotting (IB) where HCT116 (p53 Wt and p53 Null ) cell lines were treated with USP7 inhibitor P5091 (20µM) for 24 hrs. GAPDH was kept as loading control. C, Following treatment of HCT116 (p53 Null ) cells with P5091 in different doses (1, 5, 10µM) for 24 hrs, the increase in caspase 3/7 activity was determined using a fluorescence plate reader. Data represent mean ± SD of three independent biological replicates. D, Number of colonies formed by HCT116 (p53 Wt ), HepG2 (p53 Null ), and Huh7 (p53 Mut ) cells after treatment with P5091 (20 µM) for 24 hrs; colonies were counted after 15 days. E & F, Identification of USP7 and XIAP interacting proteins by Mass Spectrometry. Silver stained SDS-PAGE gels containing elute from respective pull-down as indicated. MS analysis identifies specific peptides of XIAP and USP7 respectively from GST-USP7 and GST-XIAP pull-down lanes by using HEK cell lysates. G, Identification of USP7 target proteome in HCT116 cells by label-free comparative proteomics analysis upon USP7 inhibition with P5091 (15µM for 24 hrs), figure represents the workflow of label-free quantitation (LTQ) by nano-LC−MS/M.S. on a Q-exactive followed by SIEVE TM processing. H, Graphical representation of calculated protein ratios of P5091 treated samples with respect to the vehicle control (left panel). Venn diagram showing identified number of Up-regulated, Unchanged and Down regulated proteins in HCT116 p53 wt and HCT116 p53 null cell lines upon P5091 treatment with respect to control (right panel). I & J, Whole-cell lysates were prepared from MCF7, MD-AMB 231, MD-AMB 468, T47D, C6, U87, LN18, HCT116 (p53 Wt ), HCT116 (p53 Null ), SW480, SW620, HeLa, SiHa, LnCap, A549, RAW, HEK293T, and HEK293 cells. IB analysis was performed using respective antibodies for USP7, XIAP and β-Actin. Normalized values were plotted to show a strong positive correlation between USP7 and XIAP where p value < 0.0001, n=2. Error bars in all the indicated sub-figures represent mean ± SD from three independent biological repeats. Indicated P-values were calculated using Student’s t-test and P<0.05 is represented as *, otherwise non-significant (ns).

Article Snippet: USP7 expression vector pCI-neo-Flag backbone (Addgene Plasmid # 16655).

Techniques: MTT Assay, Western Blot, Control, Activity Assay, Fluorescence, Mass Spectrometry, Staining, SDS Page, Inhibition, Quantitation Assay, Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: Identification and validation of E3 ubiquitin ligase XIAP as a novel substrate of deubiquitinase USP7 (HAUSP) - Implication towards oncogenesis

doi: 10.1101/2021.08.12.456108

Figure Lengend Snippet: A, HEK cells transfected with 2µG of EV (pGZ) (lane-1&3), pGZ-USP7 (lane-2), and consecutively inactive form of USP7 mutant pGZ-USP7 C223S (lane-4); 48 hrs post-transfection, USP7 and XIAP protein levels were checked by IB. Additionally, three different shRNAs targeting USP7 and scramble shRNA were transfected (lanes-5,6,7&8) and the levels of XIAP and USP7 were analyzed by IB. GAPDH was kept as loading control. B, qRT-PCR analysis showing relative mRNA fold change upon USP7 inhibition by P5091 (15µM) for 24 hrs. Relative mRNA fold change of XIAP upon USP7 knockdown (using siUSP7) and USP7 over-expression. Protein expression of XIAP from the same experiment was also represented by IB, where GAPDH was used as an internal loading control. C, Multiple cancer cell lines such as HCT116, C6, SH-SY5Y, SiHa, and SW620 were treated with 15µM of USP7 inhibitor (P5091-lane 2, 4, 6, 8, 10; P22077-lane 11) for 24 hrs and XIAP was analyzed by IB and compared with respective control lanes (lane 1, 3, 5, 7, 9). IB analysis of XIAP protein level in HCT116 cells transfected with GFP-USP7 (4 µG, lane 13), pGZ-USP7 C223S (4 µG, lane 14), or siUSP7 (lane 16). EV (lane 12), control Si (lane 15) were kept for control. D, Huh7 and HT29 cells were treated with USP7 inhibitor P22077 in a dose-dependent manner (5, 10, 15, 20 µM) for 24 hrs. XIAP was checked by IB. GAPDH was kept as an internal loading control. E, HCT116 cells were transfected with either Control Si (upper left panel) or siUSP7 (Upper right panel) and HEK cells were transfected with either EV (Lower left panel) or GFP-USP7 (Lower right panel) before treatment with cycloheximide (CHX: 50µG/ml). XIAP protein levels at indicated time points were analyzed by IB as shown in the figure (left panels). Graph showing the change in XIAP half-life was determined in USP7 knockdown and overexpression conditions. Error bars in all the indicated sub-figures represent mean (±) SD from three independent biological repeats. Indicated P-values were calculated using Student’s t-test and P<0.05 is represented as *, otherwise non-significant (ns).

Article Snippet: USP7 expression vector pCI-neo-Flag backbone (Addgene Plasmid # 16655).

Techniques: Transfection, Mutagenesis, shRNA, Control, Quantitative RT-PCR, Inhibition, Knockdown, Over Expression, Expressing

Journal: bioRxiv

Article Title: Identification and validation of E3 ubiquitin ligase XIAP as a novel substrate of deubiquitinase USP7 (HAUSP) - Implication towards oncogenesis

doi: 10.1101/2021.08.12.456108

Figure Lengend Snippet: A , Confocal images of HEK cells showing stains for USP7 (green), XIAP (red), and USP7-XIAP merged (yellow). Cells were counterstained with DAPI (blue). Images were captured at 60X optical magnification. B, Purified Myc-His USP7 incubated separately with purified GST and GST-XIAP in the presence of GST-Bead for 4 hrs. Proteins were separated by SDS-PAGE and analyzed by Coomassie blue staining to visualize the specific bands for GST (lane 1), GST-XIAP and Myc-His USP7 (lane 2). Figure shows the representative data of three biological replicates. C, HEK cell lysate was incubated separately with purified GST and GST-XIAP in the presence of GST-bead for 4 hrs, followed by IB using anti-USP7 and GST antibody. D, HEK cell lysate was immunoprecipitated with XIAP antibody followed by IB using antibodies against USP7 and XIAP to identify their interaction at the endogenous condition. E, HEK cells were transiently transfected with FLAG-USP7 and GFP-XIAP. Equal amounts of cell lysates were used for immunoprecipitation with Flag antibody and Normal Rabbit Serum, followed by IB using antibodies against Flag and GFP to identify their interaction under overexpressed conditions. F, HEK cells were treated with either DMSO or MG132 (25µM) for 8 hrs. Equal amounts of lysates were used for IP using USP7 antibody followed by IB using indicated antibodies to show an increased interaction of USP7 and XIAP upon blocking the proteasomal system. Veriblot secondary antibody was used to prevent nonspecific detection of the heavy and light chains. G, Workflow of domain wise docking of USP7 and XIAP proteins using three independent Docking software to identify the probable domains responsible for interaction. H, Venn diagram showing domain pair of USP7 and XIAP by using three docking softwares and identified CAT-BIR2 and UBL-BIR3 as the probable interacting domains.

Article Snippet: USP7 expression vector pCI-neo-Flag backbone (Addgene Plasmid # 16655).

Techniques: Purification, Incubation, SDS Page, Staining, Immunoprecipitation, Transfection, Blocking Assay, Software

Journal: bioRxiv

Article Title: Identification and validation of E3 ubiquitin ligase XIAP as a novel substrate of deubiquitinase USP7 (HAUSP) - Implication towards oncogenesis

doi: 10.1101/2021.08.12.456108

Figure Lengend Snippet: A, Workflow representing generation of full-length USP7 and XIAP followed by docking with Swarmdock and simulation of docking complex to identify the stable orientation of USP7 and XIAP interaction. B, Upper panels showing full-length USP7 and XIAP proteins generated by stitching the available USP7 and XIAP domains in PDB. Lower panels showing USP7-XIAP complex, where interface region highlighted in yellow. C, Schematic representation of USP7 deletion mutants and their interactions with full-length XIAP protein. D, HEK cells were co-transfected with indicated plasmid constructs; prepared the cell lysates followed by pull-down with Ni-NTA beads for 1 hr at room temperature. The pulled-down proteins and input were analyzed by IB with the indicated antibodies. Ponceau S staining indicates an equal loading in input lanes. HEK cells were transfected with the indicated plasmid constructs before the preparation of cell lysates. E, Equal amounts of lysate from plates transiently transfected as indicated, were incubated separately with purified proteins GST (right panel) or GST-XIAP fusion protein (left panel) including glutathione beads followed by IB analysis using indicated antibodies. The experiment performed thrice for biological replicates. F, Schematic representation of XIAP deletion mutants and their interaction with full-length USP7 protein. G, HEK cells were transiently transfected with Flag-USP7. The lysate was prepared and incubated separately with purified different GST tagged XIAP deletion mutants and GST protein (Control) overnight at 4°C. After pull-down with GST-bead for 2 hrs at RT, pulled-down proteins were analyzed by IB.

Article Snippet: USP7 expression vector pCI-neo-Flag backbone (Addgene Plasmid # 16655).

Techniques: Generated, Transfection, Plasmid Preparation, Construct, Staining, Incubation, Purification, Control

Journal: bioRxiv

Article Title: Identification and validation of E3 ubiquitin ligase XIAP as a novel substrate of deubiquitinase USP7 (HAUSP) - Implication towards oncogenesis

doi: 10.1101/2021.08.12.456108

Figure Lengend Snippet: HEK cells were used in experiments A-E, H & L and mentioned in other cases. A, Cells co-transfected with HA-Ub and GFP-XIAP and 24 hrs post-transfection cells were further treated with MG-132 (25µM) and vehicle control for an additional 4 hrs. GFP-tagged proteins were pulled down from lysates using GFP antibody and Protein-A Sepharose bead; analyzed by IB using indicated antibodies. B, Cells were transfected with indicated constructs; 24 hrs post-transfection, cells were treated with P5091 (15µM) for another 24 hrs. Before harvesting, the cells were further treated with MG-132 for 4 hrs. Lysates were prepared and used for pull-down using GFP antibody followed by IB using indicated antibodies. C, Cells were transfected individually with EV, USP7, and USP7 C223S plasmids. Pull-down assay was performed using XIAP antibody, bead-bound proteins and total cell lysate were analyzed by IB to detect the change in poly-ubiquitination pattern. D, Cells were transfected with indicated plasmids; 24 hr post-transfection, treated with P5091 (15µM) and vehicle control for another 24 hours. Cells were harvested after MG-132 treatment for further 4 hrs, followed by pull-down assay was performed using GFP antibody and IB with indicated antibodies. E, Cells were transfected with either WT or different Ub mutants as depicted in the figure. Following lysate preparation, pulled down the proteins using GFP antibody followed by IB using the indicated antibodies. Input (3%) was run separately for Control. F, Cell lysates prepared from p53 WT and p53 null HCT116 cells subjected to IB with the indicated antibodies. G, HCT116 (p53 wt ) cells were treated with Nutlin3A (5µM) for 24 hrs, the protein level of XIAP and other p53 responsive genes were analyzed by IB. H, Cells were treated with Nutlin3A in a dose-dependent manner (5µM and 10µM) to analyze the level of indicated proteins by IB. I, HCT116 (p53 wt ) and HT29 (p53 mut ) cells were treated with P5091 in a dose-dependent manner (0, 10, and 20µM) for 24 hrs and check the levels of the indicated proteins by IB. J, Expression of indicated genes in HCT116 cells containing either p53 wt or p53 mut was analyzed by qRT-PCR. K, HCT116 cells were treated with Doxorubicin (10µM) for 3 hrs, washed and kept in fresh media without Dox for another 21hrs before harvesting them. Expression (mRNA level) of the indicated genes was analyzed by qRT-PCR. L, Cells were transfected with either EV (left panel) or GFP-USP7 (right panel) for 24 hrs followed by Dox treatment in a dose-dependent manner (0, 1, 2, 3µM) for another 24 hrs. Expression of the indicated proteins was analyzed by IB. Expression of XIAP was normalized against GAPDH and plotted here. The data are representative of three biological replicates. qRT-PCR data represents the mean ± SD of three independent biological replicates. Indicated P-values were calculated using Student’s t-test and P<0.0001 is represented as *, otherwise non-significant (ns).

Article Snippet: USP7 expression vector pCI-neo-Flag backbone (Addgene Plasmid # 16655).

Techniques: Transfection, Control, Construct, Pull Down Assay, Ubiquitin Proteomics, Expressing, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Identification and validation of E3 ubiquitin ligase XIAP as a novel substrate of deubiquitinase USP7 (HAUSP) - Implication towards oncogenesis

doi: 10.1101/2021.08.12.456108

Figure Lengend Snippet: A, HCT 116 (p53 Wt ) cells were transfected with indicated plasmids and treated with Doxorubicin for 24 hrs to induced apoptosis, prepared the lysates and analyzed by IB using the panel of indicated antibodies. B, Similarly, HEK cells were transfected with indicated plasmids and treated with Doxorubicin (1 µM) for 24hrs to induced apoptosis, prepared the lysates and analyzed by IB using the panel of indicated antibodies. C, Huh7 (p53 Mut ) and HepG2 cells (p53 Wt ) cells were transfected with Flag-USP7 and GFP-XIAP for the indicated time points, prepared the lysates and analyzed by IB. D, HCT116 (p53 Null ) cells were transfected with either Flag-USP7 or GFP-XIAP. After 48 hrs, cells were treated with P5091 (15µM) or Doxorubicin (1µM). Prepared the cell lysates and analyzed by IB using the indicated antibodies. XIAP, USP7, and Cleaved Caspase 3 were plotted after normalization against GAPDH. E, HCT116 (p53 Null - upper panel) and HCT116 (p53 Wt - lower panel) cells were treated with either XIAP inhibitor Embellin or USP7 inhibitor P5091 for the indicated doses. Prepared the lysates and analyzed by IB. XIAP expression was plotted after normalization against GAPDH. F, HEK and C6 cells were seeded in a 6-well plate and treated with P5091 (15µM and 20 µM for HEK and, 15 µM for C6) for 24 hrs. Treated cells were stained as per protocol to detect TUNNEL positivity. G, Cell cycle profile of HCT116 (p53 WT ) cells were analyzed as per protocol after treatment with an increasing dose of P5091 (5µM, 10µM and 15µM 24 hrs). H, Scratch assay was performed to evaluate the migration of HepG2 (p53 Wt ) and Huh7 (p53 Mut ) cells pre-treated with P5091 (20µM). I, percent of cell population in different apoptosis phases of HCT116 (p53 WT ) cells were analyzed as per protocol after treatment with indicated dose of P5091 for 24 hrs. Error bars in all the indicated sub-figures represent mean (±) SD from three independent biological repeats. Indicated P-values were calculated using Student’s t-test and P<0.0001 is represented as *, otherwise non-significant (ns).

Article Snippet: USP7 expression vector pCI-neo-Flag backbone (Addgene Plasmid # 16655).

Techniques: Transfection, Expressing, Staining, Wound Healing Assay, Migration

Journal: bioRxiv

Article Title: Identification and validation of E3 ubiquitin ligase XIAP as a novel substrate of deubiquitinase USP7 (HAUSP) - Implication towards oncogenesis

doi: 10.1101/2021.08.12.456108

Figure Lengend Snippet: USP7 deubiquitinates and stabilizes anti-apoptotic protein XIAP and promote cancer cell survival. Overexpression of either USP7 or XIAP confers chemotherapy resistance and associated with increased survival of cancer cells. USP7 inhibition by small molecule inhibitor acts as a potential therapeutic intervention to fight against both p53 Wt and p53 Mut/Null cancers through MDM2 and XIAP respectively.

Article Snippet: USP7 expression vector pCI-neo-Flag backbone (Addgene Plasmid # 16655).

Techniques: Over Expression, Inhibition

Journal: iScience

Article Title: Role of the DUB enzyme USP7 in dendritic arborization, neuronal migration, and autistic-like behaviors in mice

doi: 10.1016/j.isci.2022.104595

Figure Lengend Snippet: Expression and subcellular distribution of USP7 in neurons (A) Immunostaining of USP7 and GFAP in rat hippocampal cultures at DIV15. Scale bar = 50 μm. (B) Immunostaining of USP7 and GAD67 in rat hippocampal cultures at DIV15. (C) Lysates of cultured neurons were collected on DIV15, and USP7 levels were measured by Westerns. GAPDH was probed as a loading control. (D) Lysates of different brain regions were collected from rats of embryonic day 18. USP7 levels were measured by Western blot. (E) Cultured neurons were treated with USP7 inhibitor HBX41108 (10 μM) for 2 or 4 h at DIV15, and the lysates were probed for ubiquitination. (F) Quantification showed an increase in ubiquitination intensity in the HBX41108 treated group (F(2,9) = 33.13, p < 0.01, One-way ANOVA). (G) Developmental time course of USP7 expression in the brain. Cortical tissues were collected from mice of ages from E10 to P90. Data are represented as mean ± SEM. Error bars represent SEM, ∗∗p < 0.01.

Article Snippet: Full length USP7 fragment from pCI-neo Flag HAUSP/USP7 (addgene, #16655) were inserted into the AAV-ReaChR-citrine vector.

Techniques: Expressing, Immunostaining, Cell Culture, Control, Western Blot, Ubiquitin Proteomics

Journal: iScience

Article Title: Role of the DUB enzyme USP7 in dendritic arborization, neuronal migration, and autistic-like behaviors in mice

doi: 10.1016/j.isci.2022.104595

Figure Lengend Snippet: USP7 regulates dendritic growth and arborization (A–D) Hippocampal neurons were transfected with a control vector or USP7 plasmid at DIV 7, and imaged for morphology at DIV 11 (A). Scale bar = 100 μm. Dendritic arborization was analyzed by Sholl analysis (F(1,56) = 3.865, p = 0.054, Repeated measures ANOVA) (B). The total number of dendritic branches and the sum length of dendrites showed no significant difference between the control (n = 24) and the USP7 group (n = 28) at DIV11 (p > 0.05, t -test) (C and D). (E–H) Neurons were transfected with USP7 or a vector as control at DIV 11, and imaged for morphology at DIV 15 (E). Scale bar = 100 μm. Dendritic arborization was analyzed by sholl analysis (F(1,44) = 13.037, p = 0.001, Repeated measure ANOVA) (F). The total number of dendrites and total length of dendrites were increased in USP7-transfected neurons on DIV15 (Ctrl: n = 18; USP7: n = 22. Number of dendrites, p < 0.01, t -test; sum length of dendrites, p < 0.01, t -test) (G, H). Scale bar = 100 μm. (I–M) Knockdown of USP7 results in a reduction of dendritic arborization. (I) Cortical neurons were transfected with vector (Control, n = 16) or shUSP7 (n = 31) at DIV 11 and imaged for morphology on DIV 15. Scale bar = 100 μm. (J) Sholl analysis of dendritic arborization at DIV 15 (F(1,46) = 7.497, p = 0.009, Repeated measure ANOVA). (K and L) Total number of dendrites and total length of dendrites were decreased in shUSP7 neurons on DIV15 (Number of dendrites, p < 0.05, t -test; sum length of dendrites, p < 0.01, t -test). (M) Mean length of dendrites was decreased in shUSP7 neurons on DIV15 (p < 0.05, t -test). Data are represented as mean ± SEM. Error bars represent SEM, ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: Full length USP7 fragment from pCI-neo Flag HAUSP/USP7 (addgene, #16655) were inserted into the AAV-ReaChR-citrine vector.

Techniques: Transfection, Control, Plasmid Preparation, Knockdown

Journal: iScience

Article Title: Role of the DUB enzyme USP7 in dendritic arborization, neuronal migration, and autistic-like behaviors in mice

doi: 10.1016/j.isci.2022.104595

Figure Lengend Snippet: Caspase-3 and microtubules are the downstream effectors mediating the USP7 effect on dendritic arborization (A) Primary hippocampal neurons were transfected with USP7 and immunostained for the cleaved caspase 3. Arrows indicate the transfected neurons. Scale bar = 50 μm. (B) Quantification showed a decrease in cleaved caspase-3 intensity in USP7 overexpressing neurons (Ctrl: n = 15; USP7: n = 19, t -test). (C) HEK cells were transfected with USP7 and shUSP7. Cell lysates were collected to determine the cleaved caspase-3 levels by Westerns. (D) Quantification of Western blot intensities of cleaved caspase-3 (F(2,6) = 16.56, p < 0.01, one-way ANOVA, Tukey). (E) Primary neurons were infected with AAV-USP7 at DIV 0, and neuron lysates were collected for western blot to detect changes in microtubule cleavage. (F) Quantification showed a significant decrease in cleaved microtubules in neurons with USP7 virus infection (Ctrl: n = 3; USP7: n = 3, t -test). (G) Primary neurons were infected with AAV-USP7 at DIV 0 and immunostained at DIV 15 with TubΔCasp6 antibodies. Scale bar = 50 μm. (H and I) Quantification showed that USP7-infected neurons had a decrease in microtubule cleavage intensity (Ctrl: n = 15; USP7: n = 18, t -test), but not in the number of cleavage sites along the dendrite (Ctrl: n = 20; USP7: n = 18, t -test). Data are represented as mean ± SEM. Error bars represent SEM, ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: Full length USP7 fragment from pCI-neo Flag HAUSP/USP7 (addgene, #16655) were inserted into the AAV-ReaChR-citrine vector.

Techniques: Transfection, Western Blot, Infection, Virus

Journal: iScience

Article Title: Role of the DUB enzyme USP7 in dendritic arborization, neuronal migration, and autistic-like behaviors in mice

doi: 10.1016/j.isci.2022.104595

Figure Lengend Snippet: USP7 regulates XIAP protein accumulation in neurons (A) Lysates were collected from primary cortical neurons of DIV 0 to DIV 20. Expression levels of USP7 and XIAP were measured by Western blot. GAPDH was probed as a loading control. (B) Quantification of USP7 and XIAP intensity (n = 3). (C–E) Cortical neurons were infected with AAV-GFP or AAV-USP7 on DIV 0 for 15 days, and USP7 and XIAP levels were measured by Western blot. An increased level for both USP7 and XIAP was detected in neurons infected with USP7 virus (GFP-AAV: n = 4; USP7-AAV: n = 4. p < 0.05, t -test). (F and G) Cortical neurons were treated with HBX41108 (USP7 inhibitor) for 2 and 4 h and the lysates were collected to probe for XIAP. Inhibition of USP7 led to a decrease in XIAP amount. (Ctrl: n = 4; HBX 2 h: n = 4; HBX 4 h: n = 4. F(2,9) = 30.88, p < 0.01, one-way ANOVA, Dunnett). (H) Cortical neurons were transfected with USP7 at DIV 11 and immunostained for XIAP at DIV 15. Scale bar = 50 μm. (I) Quantification showed an increase in endogenous XIAP intensity in neurons transfected with USP7 (Ctrl: n = 13; USP7: n = 13. p < 0.01, t -test). (J) Hippocampal neurons were transfected with shUSP7 at DIV 8 and immunostained for XIAP at DIV 15. Scale bar = 50 μm. (K) Quantification showed a decrease in endogenous XIAP in neurons with USP7 knockdown (Ctrl: n = 14; USP7: n = 14. p < 0.01, t -test). Data are represented as mean ± SEM. Error bars represent SEM, ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: Full length USP7 fragment from pCI-neo Flag HAUSP/USP7 (addgene, #16655) were inserted into the AAV-ReaChR-citrine vector.

Techniques: Expressing, Western Blot, Control, Infection, Virus, Inhibition, Transfection, Knockdown

Journal: iScience

Article Title: Role of the DUB enzyme USP7 in dendritic arborization, neuronal migration, and autistic-like behaviors in mice

doi: 10.1016/j.isci.2022.104595

Figure Lengend Snippet: USP7 causes XIAP deubiquitination and stabilization (A) XIAP ubiquitination assay. HEK293 cells were transfected with FLAG-XIAP, HA-ubiquitin, and USP7 for 2 days. XIAP was immunoprecipitated and probed for ubiquitin (ubi). Cell lysates (input) were used to detect protein levels. (B and C) Quantification of Western blot intensities. USP7 caused a decrease in XIAP ubiquitination and a decrease in XIAP protein levels (Ubiquitination Signal: F(2,9) = 20.95, p < 0.01, one-way ANOVA, Tukey; XIAP Signal: F(2,9) = 22.20, p < 0.01, one-way ANOVA, Tukey. XIAP: n = 4; XIAP + Ubi: n = 4; XIAP + Ubi + USP7: n = 4). (D) Degradation assay of XIAP with or without USP7. Transfected HEK cells were treated with cycloheximide (CHX) for various time periods and cell lysates were collected to examine XIAP levels by Western blot. (E) Quantification of the degradation rate of XIAP over time (Treatment: F(1,4) = 10.14, p < 0.05, repeated measure ANOVA). (F) Morphology of primary neurons transfected with USP7 or XIAP alone, or both. Scale bar = 100 μm. (G and H) Dendrite branch number and the total length of dendrite were increased in neurons overexpressing USP7 or XIAP. Co-transfection of USP7 and XIAP had no additional effects compared with USP7 alone or XIAP only group (Ctrl: n = 35; USP7: n = 25; XIAP: n = 34; XIAP + USP7: n = 31. Number of dendrites: F(3,121) = 13.83, p < 0.01, one-way ANOVA, Tukey; Sum dendrite length: F(3,121) = 8.82, p < 0.01. one-way ANOVA, Tukey). Data are represented as mean ± SEM. Error bars represent SEM, ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: Full length USP7 fragment from pCI-neo Flag HAUSP/USP7 (addgene, #16655) were inserted into the AAV-ReaChR-citrine vector.

Techniques: Ubiquitin Proteomics, Transfection, Immunoprecipitation, Western Blot, Degradation Assay, Cotransfection

Journal: iScience

Article Title: Role of the DUB enzyme USP7 in dendritic arborization, neuronal migration, and autistic-like behaviors in mice

doi: 10.1016/j.isci.2022.104595

Figure Lengend Snippet: Knockdown of XIAP blocks the effect of USP7 on dendritic arborization (A) Cultured neurons were transfected with shXIAP at DIV 7 and immunostained for XIAP at DIV 11. Scale bar = 50 μm. (B) Quantification of XIAP expression (Ctrl: n = 36; shXIAP: n = 36. p < 0.01, t -test). (C) Morphology of primary neurons transfected with USP7 or shXIAP alone, or both at DIV11. Scale bar = 100 μm. (D and E) Dendrite branch number and the total length of dendrite were decreased in XIAP knockdown neurons. Co-transfection of USP7 and shXIAP had no additional effects compared with shXIAP alone group, but decreased significantly compared with USP7 only group (Ctrl: n = 35; USP7: n = 46; shXIAP: n = 46; USP7+shXIAP: n = 35. Number of dendrites: F(3,148) = 10.69, p < 0.01, one-way ANOVA, Tukey; Sum dendrite length: F(3,148) = 10.01, p < 0.01. one-way ANOVA, Tukey). (F) Sholl analysis of dendritic arborization at DIV 11 (Group: F (3, 154) = 11.23, p < 0.01. Ctrl vs. shXIAP: p < 0.01; Ctrl vs. USP7: p > 0.05; USP7+shXIAP vs. USP7: p < 0.01; USP7+shXIAP vs. shXIAP: p > 0.05. Repeated measures ANOVA, Tukey). Data are represented as mean ± SEM. Error bars represent SEM, ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: Full length USP7 fragment from pCI-neo Flag HAUSP/USP7 (addgene, #16655) were inserted into the AAV-ReaChR-citrine vector.

Techniques: Knockdown, Cell Culture, Transfection, Expressing, Cotransfection

Journal: iScience

Article Title: Role of the DUB enzyme USP7 in dendritic arborization, neuronal migration, and autistic-like behaviors in mice

doi: 10.1016/j.isci.2022.104595

Figure Lengend Snippet: USP7 antagonizes E6AP effect on dendritic arborization via control of XIAP ubiquitination (A) HEK293 cells were transfected with FLAG-XIAP, HA-ubiquitin, and USP7 with or without E6AP for 2 d. XIAP was immunoprecipitated and probed for HA-ubiquitin (HA-ubi). Cell lysates (input) were also probed to detect the total protein levels. (B and C) Quantification of western blot intensities. E6AP caused an increase in XIAP ubiquitination (F(3,12) = 19.62, p < 0.01, one-way ANOVA, Tukey) and a decrease in XIAP protein levels in the input (F(3,8) = 43.69, p < 0.01, one-way ANOVA, Tukey). USP7 blocked the effect of E6AP on XIAP protein accumulation and XIAP ubiquitination. (D) Morphology of primary neurons transfected with USP7, E6AP, and USP7+E6AP (DIV 11 - DIV 15). Scale bar = 100 μm. (E and F) Dendritic branch number (F(3,81) = 17.28, p < 0.01, one-way ANOVA, Tukey) and the total length of dendrites (F(3,81) = 17.92, p < 0.01, one-way ANOVA, Tukey) were decreased in neurons with E6AP overexpression (E6AP: n = 19; Ctrl: n = 20. one-way ANOVA, Tukey). USP7 abolished the effect caused by E6AP expression (E6AP + USP7: n = 21; E6AP: n = 19. one-way ANOVA, Tukey). (G) Sholl analysis showed a reduction in the complexity of dendritic arborization in E6AP neurons, which was blocked by co-expression with USP7 in neurons (Group: F(3,77) = 16.833, p < 0.01. Repeated measures ANOVA, Tukey). Data are represented as mean ± SEM. Error bars represent SEM, ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: Full length USP7 fragment from pCI-neo Flag HAUSP/USP7 (addgene, #16655) were inserted into the AAV-ReaChR-citrine vector.

Techniques: Control, Ubiquitin Proteomics, Transfection, Immunoprecipitation, Western Blot, Over Expression, Expressing

Journal: iScience

Article Title: Role of the DUB enzyme USP7 in dendritic arborization, neuronal migration, and autistic-like behaviors in mice

doi: 10.1016/j.isci.2022.104595

Figure Lengend Snippet: USP7 overexpression in the brain affects neuron migration and dendritic arborization (A) Schematic illustration of the procedures for in utero electroporation (IUE) performed at E15 and P0, P15. (B) Brain slices taken at P0 following IUE of DsRed control (Ctrl) or GFP-USP7 at E15. Scale bar = 200 μm. (C) Analysis of neuronal migration at P0 showed that less neurons were distributed in the upCP and more in the loCP and VZ regions compared with controls (Ctrl: n = 12; USP7: n = 14. t -test). More than 1200 GFP + neurons from four brains were analyzed in each group. 15 slices from five brains were analyzed in each group, and more than 3000 GFP + neurons were analyzed totally. (D) Representative images showing dendritic arborizations of control and USP7 groups. Scale bar = 50 μm. (E) Sholl analysis of dendritic structure at P15 after IUE showed a significant change in branching (Group: F(1, 48) = 17.06, p < 0.01, Repeated measures ANOVA. Ctrl: n = 27; USP7: n = 22). (F and G) Dendrite numbers and the total length of dendrites were increased in the USP7 overexpressing group compared with the control (Ctrl: n = 27; USP7: n = 22. t -test). Data are represented as mean ± SEM. Error bars represent SEM, ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: Full length USP7 fragment from pCI-neo Flag HAUSP/USP7 (addgene, #16655) were inserted into the AAV-ReaChR-citrine vector.

Techniques: Over Expression, Migration, In Utero, Electroporation, Control

Journal: iScience

Article Title: Role of the DUB enzyme USP7 in dendritic arborization, neuronal migration, and autistic-like behaviors in mice

doi: 10.1016/j.isci.2022.104595

Figure Lengend Snippet: USP7 overexpression in vivo in mouse brain at P0 leads to dendrite morphological changes (A) Schematic illustration of the procedures for virus injection at P0 and schedule for behavior tests performed at P30-P55. AAV-USP7 and AAV-GFP were injected into the lateral ventricles in mice at P0, and the mice were perfused around P30 to P60 for cryostat to show the validity of the virus. The expression of GFP (B) or GFP-fused USP7 (left: Scale bar = 1000 μm; right: Scale bar = 200 μm) (C) can be detected in the whole brain around P30 to P60 ( left: Scale bar = 500 μm; right: Scale bar = 100 μm). (D) Western blot showed that the expression of USP7 in the cortex of the AAV USP7 group is significantly higher than in the control group, as well as XIAP. (E–I) Sholl analysis shows the morphological changes. Scale bar = 50 μm. 19 cortical neurons from 5 control mice and 27 neurons from 5 AAV USP7 mice were analyzed. The number of dendrites and the mean and total length of dendrites were all increased significantly in the AAV USP7 group (F, Number of dendrites: Ctrl: n = 19, USP7: n = 27, p < 0.01, t -test; G,Sum length: Ctrl: n = 19, USP7: n = 27, p < 0.01, t -test; H, Mean length: Ctrl: n = 19, USP7: n = 27, p < 0.01. t -test). I, The dendritic arborization was more complex in the AAV USP7 group compared with the control group (Group: F(1, 44) = 37.91, p < 0.01, Repeated measures ANOVA). Data are represented as mean ± SEM. Error bars represent SEM, ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: Full length USP7 fragment from pCI-neo Flag HAUSP/USP7 (addgene, #16655) were inserted into the AAV-ReaChR-citrine vector.

Techniques: Over Expression, In Vivo, Virus, Injection, Expressing, Western Blot, Control

Journal: iScience

Article Title: Role of the DUB enzyme USP7 in dendritic arborization, neuronal migration, and autistic-like behaviors in mice

doi: 10.1016/j.isci.2022.104595

Figure Lengend Snippet: Behavior changes in mice following USP7 overexpression in the brain at P0. Behavioral tests were performed at P30-P55 (A and B) Homecage activities including grooming, rearing, digging, climbing, circling, and jumping (Ctrl: n = 9; USP7: n = 13, t -test). Counts of grooming and digging (A), as well as the overall activity events (B), were increased significantly in USP7 infected mice. (C) Track length in the open field test showed no difference between two groups ( t -test). (D) A representative example of the test arena at the end of the Marble burying test. (E) USP7 mice buried more marbles during the test (F(1,20) = 13.46, p < 0.01. Ctrl: n = 9; USP7: n = 13, repeated measures ANOVA). (F) Quantification of the number of marbles buried at the end of the test (30 min). (G and J) Paradigm for the social preference test (G) and social novelty test (J), and representative tracing. (H and K) Quantification of time spent in each chamber in the social preference test (H) (Ctrl: n = 8; USP7: n = 12, t -test) and social novelty test (K) (Ctrl: n = 8; USP7: n = 12, t -test). (I and L) Quantification of the preference index in the social preference test (I) and social novelty test (L). The preference for social interaction was increased in USP7 animals compared with the control ( t -test) (I). (M and N) The novel object recognition test showed no difference in discrimination index between the two groups (Ctrl: n = 8; USP7: n = 13, t -test). (O and P) Hot plate test. The withdrawal latency was decreased in USP7 mice compared with the control (Ctrl: n = 8; USP7: n = 13, t -test). Data are represented as mean ± SEM. Error bars represent SEM, ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: Full length USP7 fragment from pCI-neo Flag HAUSP/USP7 (addgene, #16655) were inserted into the AAV-ReaChR-citrine vector.

Techniques: Over Expression, Activity Assay, Infection, Control, Hot Plate Test

Journal: iScience

Article Title: Role of the DUB enzyme USP7 in dendritic arborization, neuronal migration, and autistic-like behaviors in mice

doi: 10.1016/j.isci.2022.104595

Figure Lengend Snippet:

Article Snippet: Full length USP7 fragment from pCI-neo Flag HAUSP/USP7 (addgene, #16655) were inserted into the AAV-ReaChR-citrine vector.

Techniques: Virus, Transfection, Recombinant, Sequencing, Software