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: Figure 1. Expression and subcellular distribution of USP7 in neurons (A) Immunostaining of USP7 and GFAP in rat hippocampal cultures at DIV15. Scale bar = 50 mm. (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 mM) 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 G SEM. Error bars represent SEM, **p < 0.01.

Article Snippet: Viral constructs preparation and virus infection Full length USP7 fragment from pCI-neo Flag HAUSP/USP7 (addgene, #16655) were inserted into the AAVReaChR-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: Figure 2. 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 mm. 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 mm. 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 mm. (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 mm.

Article Snippet: Viral constructs preparation and virus infection Full length USP7 fragment from pCI-neo Flag HAUSP/USP7 (addgene, #16655) were inserted into the AAVReaChR-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: Figure 3. 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 mm. (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 mm. (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 G SEM. Error bars represent SEM, *p < 0.05, **p < 0.01.

Article Snippet: Viral constructs preparation and virus infection Full length USP7 fragment from pCI-neo Flag HAUSP/USP7 (addgene, #16655) were inserted into the AAVReaChR-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: Figure 4. 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 mm. (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 G SEM. Error bars represent SEM, *p < 0.05, **p < 0.01.

Article Snippet: Viral constructs preparation and virus infection Full length USP7 fragment from pCI-neo Flag HAUSP/USP7 (addgene, #16655) were inserted into the AAVReaChR-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: Figure 5. 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 mm. (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 mm. (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 G SEM. Error bars represent SEM, *p < 0.05, **p < 0.01.

Article Snippet: Viral constructs preparation and virus infection Full length USP7 fragment from pCI-neo Flag HAUSP/USP7 (addgene, #16655) were inserted into the AAVReaChR-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: Figure 6. 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 mm. (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 G SEM. Error bars represent SEM, *p < 0.05, **p < 0.01.

Article Snippet: Viral constructs preparation and virus infection Full length USP7 fragment from pCI-neo Flag HAUSP/USP7 (addgene, #16655) were inserted into the AAVReaChR-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: Figure 7. 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 mm. (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 TubDCasp6 antibodies. Scale bar = 50 mm. (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 G SEM. Error bars represent SEM, *p < 0.05, **p < 0.01.

Article Snippet: Viral constructs preparation and virus infection Full length USP7 fragment from pCI-neo Flag HAUSP/USP7 (addgene, #16655) were inserted into the AAVReaChR-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: Figure 8. 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 mm. (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 mm. (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 G SEM. Error bars represent SEM, *p < 0.05, **p < 0.01.

Article Snippet: Viral constructs preparation and virus infection Full length USP7 fragment from pCI-neo Flag HAUSP/USP7 (addgene, #16655) were inserted into the AAVReaChR-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: Figure 9. 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 mm; right: Scale bar = 200 mm) (C) can be detected in the whole brain around P30 to P60 ( left: Scale bar = 500 mm; right: Scale bar = 100 mm). (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 mm. 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 G SEM. Error bars represent SEM, *p < 0.05, **p < 0.01.

Article Snippet: Viral constructs preparation and virus infection Full length USP7 fragment from pCI-neo Flag HAUSP/USP7 (addgene, #16655) were inserted into the AAVReaChR-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: Figure 10. 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.

Article Snippet: Viral constructs preparation and virus infection Full length USP7 fragment from pCI-neo Flag HAUSP/USP7 (addgene, #16655) were inserted into the AAVReaChR-citrine vector.

Techniques: Over Expression, Activity Assay, Infection

Journal: Journal of Virology

Article Title: Regulation of ICP0-Null Mutant Herpes Simplex Virus Type 1 Infection by ND10 Components ATRX and hDaxx

doi: 10.1128/jvi.02597-09

Figure Lengend Snippet: FIG. 1. Analysis of endogenous expression of ND10 proteins in cultured cell lines. Samples of the indicated cells were harvested, and whole-cell extracts derived from approximately 3 104 cells were loaded onto 7.5% or 6% polyacrylamide gels. The antibodies used for Western blot detection were anti-PML 5E10, anti-ATRX 39F, anti- Daxx D7810, anti-Sp100 SpGH, anti-actin AL-40, and anti-USP7 BL851. USP7 and actin were used as loading controls for 6% and 7.5% gels, respectively. The differences in USP7 expression were reproduc- ible.

Article Snippet: Rabbit anti-USP7 antibody was from Bethyl Laboratories (BL851), while anti-USP7 rabbit serum 201 has been described previously (23, 24).

Techniques: Expressing, Cell Culture, Derivative Assay, Western Blot

Journal: Journal of Virology

Article Title: Regulation of ICP0-Null Mutant Herpes Simplex Virus Type 1 Infection by ND10 Components ATRX and hDaxx

doi: 10.1128/jvi.02597-09

Figure Lengend Snippet: FIG. 6. Efficiency of HSV-1 infection in ATRX-depleted cells. (A) Detection of ATRX expression by Western blot analysis using 39F antibody, showing expression of both ATRX isoforms in HALL cells and barely detectable levels of expression in HAA cells. Samples were resolved on a 6% polyacrylamide gel. USP7 was used as a loading control. (B) Kinetics of viral protein expression in ATRX-depleted cells. HALL and HAA cells were infected with wt or ICP0-null HSV-1 at an MOI of 2, and samples were harvested for Western blot analysis at 0, 4, 6, and 8 h postinfection (hpi). Samples were resolved on a 7.5% polyacrylamide gel, and membranes were probed for ICP4 and UL42, using antibodies 58S and Z1F11, respectively. Actin was a loading control. (C) Efficiency of plaque formation of HSV-1 in ATRX-de- pleted cells. Cells were infected with wt HSV-1 strain in1863 and ICP0-null HSV-1 strain dl1403/CMVlacZ at sequential dilutions. After 24 h of infection, cells were fixed and stained for -galactosidase expression. The plots represent the mean relative efficiencies of plaque formation as the numbers of plaques determined to occur in HAA cells in relation to the level for HALL cells from three independent exper- iments. Error bars represent standard error values.

Article Snippet: Rabbit anti-USP7 antibody was from Bethyl Laboratories (BL851), while anti-USP7 rabbit serum 201 has been described previously (23, 24).

Techniques: Infection, Expressing, Western Blot, Control, Staining

Journal: Journal of Virology

Article Title: Regulation of ICP0-Null Mutant Herpes Simplex Virus Type 1 Infection by ND10 Components ATRX and hDaxx

doi: 10.1128/jvi.02597-09

Figure Lengend Snippet: FIG. 8. Deletion of the ATRX interaction domain of hDaxx. (A) Schematic representation of wt hDaxx (based on that in reference 102), the EYFP-hDaxx construct, and the EYFP-hDaxxPAH1 mu- tant, from which amino acids 48 to 120 spanning the ATRX interacting domain PAH1 were removed by PCR-based splicing mutagenesis. PAH, paired amphipathic helices; D/E, Asp/Glu-rich motif; S/P/T, Ser/Pro/Thr-rich domain. (B) Western blot analysis of expression of hDaxx proteins in HepaRG, HALL, HD-E, HD-ED, and HD.PAH cells. The membrane was probed with the indicated antibodies. Sam- ples were resolved on a 7.5% polyacrylamide gel. (C) Immunoprecipi- tation analysis of ATRX and hDaxx interaction in HD-ED cells and HD.PAH cells. Lanes 1 and 6 represent 2.5% input of the total extract sample incubated in each immunoprecipitation reaction. HD-ED and HD.PAH cells extracts were immunoprecipitated with either anti-ATRX (antibody H300; lanes 4 and 9), anti-Daxx (antibody 07-471; lanes 5 and 10), control rabbit serum 201 raised against USP7 (lanes 3 and 8), or no antibody (lanes 2 and 7). Samples were analyzed on a 6% polyacrylamide gel and probed with anti-ATRX 39F and anti-hDaxx D7810 antibodies.

Article Snippet: Rabbit anti-USP7 antibody was from Bethyl Laboratories (BL851), while anti-USP7 rabbit serum 201 has been described previously (23, 24).

Techniques: Construct, PCR Splicing, Western Blot, Expressing, Membrane, Incubation, Immunoprecipitation, Control

Journal: Journal of Virology

Article Title: Regulation of ICP0-Null Mutant Herpes Simplex Virus Type 1 Infection by ND10 Components ATRX and hDaxx

doi: 10.1128/jvi.02597-09

Figure Lengend Snippet: FIG. 12. ICP0 neither interacts with nor disrupts the ATRX/hDaxx complex. HepaRG cells were infected with wt HSV-1 at an MOI of 5, and cells were harvested in IP lysis buffer at 4 h postinfection. Immu- noprecipitation was carried out using no antibody or anti-ICP0 190, anti-ATRX H300, anti-Daxx 07-471, and anti-USP7 210 antibodies, as indicated. Input lanes contained 2.5% of the sample used for immu- noprecipitation. Western blot analysis was carried out using mouse anti-ATRX 39F, anti-Daxx D7810, and anti-ICP0 11060 antibodies.

Article Snippet: Rabbit anti-USP7 antibody was from Bethyl Laboratories (BL851), while anti-USP7 rabbit serum 201 has been described previously (23, 24).

Techniques: Infection, Lysis, Western Blot

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: Figure 1. Expression and subcellular distribution of USP7 in neurons (A) Immunostaining of USP7 and GFAP in rat hippocampal cultures at DIV15. Scale bar = 50 mm. (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 mM) 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 G SEM. Error bars represent SEM, **p < 0.01.

Article Snippet: Antibodies mouse anti-USP7 Santa Cruz Cat# sc-137008; RRID: AB_2214163 rabbit anti-XIAP Bioss Antibodies Cat# bs-1281r; RRID: AB_10856518 rabbit anti-XIAP Sigma-Aldrich Cat# PRS3331; RRID: AB_1858883 mouse anti-FLAG tag DYKDDDDK Cell Signaling Technology Cat# 8146; RRID: AB_10950495 mouse anti-PSD95 synaptic systems Cat# 124011; RRID: AB_10804286 mouse anti-E6AP Sigma-Aldrich Cat# E8655; RRID: AB_261956 rabbit anti-cleaved caspase-3 Cell Signaling Technology Cat# 9661; RRID: AB_2341188 anti-GAPDH EMD Millipore Cat# MAB374; RRID: AB_2107445 mouse anti-a tubulin Sigma-Aldrich Cat# T9026; RRID: AB_477593 rabbit anti-cleaved tubulin (TubDCasp6) provided kindly by Andrea LeBlanc at McGill University N/A mouse anti-NeuN EMD Millipore Cat# MAB377; RRID: AB_2298772 rabbit anti-Ubiquitin Abcam Cat# ab19247; RRID: AB_444805 mouse-IgG-HRP Bio-Rad Cat# 170-6516; RRID: AB_11125547 Rabbit-IgG-HRP Bio-Rad Cat# 170-6515; RRID: AB_11125142 mouse Alexa Fluor 488 Invitrogen Cat# A21121; RRID: AB_2535764 rabbit Alexa Fluor 488 Invitrogen Cat# A11094; RRID: AB_221544 mouse Alexa Fluor 555 Invitrogen Cat# A21127; RRID: AB_141596 rabbit Alexa Fluor 555 Invitrogen Cat# A21428; RRID: AB_2535849

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: Figure 2. 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 mm. 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 mm. 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 mm. (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 mm.

Article Snippet: Antibodies mouse anti-USP7 Santa Cruz Cat# sc-137008; RRID: AB_2214163 rabbit anti-XIAP Bioss Antibodies Cat# bs-1281r; RRID: AB_10856518 rabbit anti-XIAP Sigma-Aldrich Cat# PRS3331; RRID: AB_1858883 mouse anti-FLAG tag DYKDDDDK Cell Signaling Technology Cat# 8146; RRID: AB_10950495 mouse anti-PSD95 synaptic systems Cat# 124011; RRID: AB_10804286 mouse anti-E6AP Sigma-Aldrich Cat# E8655; RRID: AB_261956 rabbit anti-cleaved caspase-3 Cell Signaling Technology Cat# 9661; RRID: AB_2341188 anti-GAPDH EMD Millipore Cat# MAB374; RRID: AB_2107445 mouse anti-a tubulin Sigma-Aldrich Cat# T9026; RRID: AB_477593 rabbit anti-cleaved tubulin (TubDCasp6) provided kindly by Andrea LeBlanc at McGill University N/A mouse anti-NeuN EMD Millipore Cat# MAB377; RRID: AB_2298772 rabbit anti-Ubiquitin Abcam Cat# ab19247; RRID: AB_444805 mouse-IgG-HRP Bio-Rad Cat# 170-6516; RRID: AB_11125547 Rabbit-IgG-HRP Bio-Rad Cat# 170-6515; RRID: AB_11125142 mouse Alexa Fluor 488 Invitrogen Cat# A21121; RRID: AB_2535764 rabbit Alexa Fluor 488 Invitrogen Cat# A11094; RRID: AB_221544 mouse Alexa Fluor 555 Invitrogen Cat# A21127; RRID: AB_141596 rabbit Alexa Fluor 555 Invitrogen Cat# A21428; RRID: AB_2535849

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: Figure 3. 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 mm. (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 mm. (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 G SEM. Error bars represent SEM, *p < 0.05, **p < 0.01.

Article Snippet: Antibodies mouse anti-USP7 Santa Cruz Cat# sc-137008; RRID: AB_2214163 rabbit anti-XIAP Bioss Antibodies Cat# bs-1281r; RRID: AB_10856518 rabbit anti-XIAP Sigma-Aldrich Cat# PRS3331; RRID: AB_1858883 mouse anti-FLAG tag DYKDDDDK Cell Signaling Technology Cat# 8146; RRID: AB_10950495 mouse anti-PSD95 synaptic systems Cat# 124011; RRID: AB_10804286 mouse anti-E6AP Sigma-Aldrich Cat# E8655; RRID: AB_261956 rabbit anti-cleaved caspase-3 Cell Signaling Technology Cat# 9661; RRID: AB_2341188 anti-GAPDH EMD Millipore Cat# MAB374; RRID: AB_2107445 mouse anti-a tubulin Sigma-Aldrich Cat# T9026; RRID: AB_477593 rabbit anti-cleaved tubulin (TubDCasp6) provided kindly by Andrea LeBlanc at McGill University N/A mouse anti-NeuN EMD Millipore Cat# MAB377; RRID: AB_2298772 rabbit anti-Ubiquitin Abcam Cat# ab19247; RRID: AB_444805 mouse-IgG-HRP Bio-Rad Cat# 170-6516; RRID: AB_11125547 Rabbit-IgG-HRP Bio-Rad Cat# 170-6515; RRID: AB_11125142 mouse Alexa Fluor 488 Invitrogen Cat# A21121; RRID: AB_2535764 rabbit Alexa Fluor 488 Invitrogen Cat# A11094; RRID: AB_221544 mouse Alexa Fluor 555 Invitrogen Cat# A21127; RRID: AB_141596 rabbit Alexa Fluor 555 Invitrogen Cat# A21428; RRID: AB_2535849

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: Figure 4. 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 mm. (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 G SEM. Error bars represent SEM, *p < 0.05, **p < 0.01.

Article Snippet: Antibodies mouse anti-USP7 Santa Cruz Cat# sc-137008; RRID: AB_2214163 rabbit anti-XIAP Bioss Antibodies Cat# bs-1281r; RRID: AB_10856518 rabbit anti-XIAP Sigma-Aldrich Cat# PRS3331; RRID: AB_1858883 mouse anti-FLAG tag DYKDDDDK Cell Signaling Technology Cat# 8146; RRID: AB_10950495 mouse anti-PSD95 synaptic systems Cat# 124011; RRID: AB_10804286 mouse anti-E6AP Sigma-Aldrich Cat# E8655; RRID: AB_261956 rabbit anti-cleaved caspase-3 Cell Signaling Technology Cat# 9661; RRID: AB_2341188 anti-GAPDH EMD Millipore Cat# MAB374; RRID: AB_2107445 mouse anti-a tubulin Sigma-Aldrich Cat# T9026; RRID: AB_477593 rabbit anti-cleaved tubulin (TubDCasp6) provided kindly by Andrea LeBlanc at McGill University N/A mouse anti-NeuN EMD Millipore Cat# MAB377; RRID: AB_2298772 rabbit anti-Ubiquitin Abcam Cat# ab19247; RRID: AB_444805 mouse-IgG-HRP Bio-Rad Cat# 170-6516; RRID: AB_11125547 Rabbit-IgG-HRP Bio-Rad Cat# 170-6515; RRID: AB_11125142 mouse Alexa Fluor 488 Invitrogen Cat# A21121; RRID: AB_2535764 rabbit Alexa Fluor 488 Invitrogen Cat# A11094; RRID: AB_221544 mouse Alexa Fluor 555 Invitrogen Cat# A21127; RRID: AB_141596 rabbit Alexa Fluor 555 Invitrogen Cat# A21428; RRID: AB_2535849

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: Figure 5. 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 mm. (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 mm. (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 G SEM. Error bars represent SEM, *p < 0.05, **p < 0.01.

Article Snippet: Antibodies mouse anti-USP7 Santa Cruz Cat# sc-137008; RRID: AB_2214163 rabbit anti-XIAP Bioss Antibodies Cat# bs-1281r; RRID: AB_10856518 rabbit anti-XIAP Sigma-Aldrich Cat# PRS3331; RRID: AB_1858883 mouse anti-FLAG tag DYKDDDDK Cell Signaling Technology Cat# 8146; RRID: AB_10950495 mouse anti-PSD95 synaptic systems Cat# 124011; RRID: AB_10804286 mouse anti-E6AP Sigma-Aldrich Cat# E8655; RRID: AB_261956 rabbit anti-cleaved caspase-3 Cell Signaling Technology Cat# 9661; RRID: AB_2341188 anti-GAPDH EMD Millipore Cat# MAB374; RRID: AB_2107445 mouse anti-a tubulin Sigma-Aldrich Cat# T9026; RRID: AB_477593 rabbit anti-cleaved tubulin (TubDCasp6) provided kindly by Andrea LeBlanc at McGill University N/A mouse anti-NeuN EMD Millipore Cat# MAB377; RRID: AB_2298772 rabbit anti-Ubiquitin Abcam Cat# ab19247; RRID: AB_444805 mouse-IgG-HRP Bio-Rad Cat# 170-6516; RRID: AB_11125547 Rabbit-IgG-HRP Bio-Rad Cat# 170-6515; RRID: AB_11125142 mouse Alexa Fluor 488 Invitrogen Cat# A21121; RRID: AB_2535764 rabbit Alexa Fluor 488 Invitrogen Cat# A11094; RRID: AB_221544 mouse Alexa Fluor 555 Invitrogen Cat# A21127; RRID: AB_141596 rabbit Alexa Fluor 555 Invitrogen Cat# A21428; RRID: AB_2535849

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: Figure 6. 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 mm. (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 G SEM. Error bars represent SEM, *p < 0.05, **p < 0.01.

Article Snippet: Antibodies mouse anti-USP7 Santa Cruz Cat# sc-137008; RRID: AB_2214163 rabbit anti-XIAP Bioss Antibodies Cat# bs-1281r; RRID: AB_10856518 rabbit anti-XIAP Sigma-Aldrich Cat# PRS3331; RRID: AB_1858883 mouse anti-FLAG tag DYKDDDDK Cell Signaling Technology Cat# 8146; RRID: AB_10950495 mouse anti-PSD95 synaptic systems Cat# 124011; RRID: AB_10804286 mouse anti-E6AP Sigma-Aldrich Cat# E8655; RRID: AB_261956 rabbit anti-cleaved caspase-3 Cell Signaling Technology Cat# 9661; RRID: AB_2341188 anti-GAPDH EMD Millipore Cat# MAB374; RRID: AB_2107445 mouse anti-a tubulin Sigma-Aldrich Cat# T9026; RRID: AB_477593 rabbit anti-cleaved tubulin (TubDCasp6) provided kindly by Andrea LeBlanc at McGill University N/A mouse anti-NeuN EMD Millipore Cat# MAB377; RRID: AB_2298772 rabbit anti-Ubiquitin Abcam Cat# ab19247; RRID: AB_444805 mouse-IgG-HRP Bio-Rad Cat# 170-6516; RRID: AB_11125547 Rabbit-IgG-HRP Bio-Rad Cat# 170-6515; RRID: AB_11125142 mouse Alexa Fluor 488 Invitrogen Cat# A21121; RRID: AB_2535764 rabbit Alexa Fluor 488 Invitrogen Cat# A11094; RRID: AB_221544 mouse Alexa Fluor 555 Invitrogen Cat# A21127; RRID: AB_141596 rabbit Alexa Fluor 555 Invitrogen Cat# A21428; RRID: AB_2535849

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: Figure 7. 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 mm. (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 TubDCasp6 antibodies. Scale bar = 50 mm. (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 G SEM. Error bars represent SEM, *p < 0.05, **p < 0.01.

Article Snippet: Antibodies mouse anti-USP7 Santa Cruz Cat# sc-137008; RRID: AB_2214163 rabbit anti-XIAP Bioss Antibodies Cat# bs-1281r; RRID: AB_10856518 rabbit anti-XIAP Sigma-Aldrich Cat# PRS3331; RRID: AB_1858883 mouse anti-FLAG tag DYKDDDDK Cell Signaling Technology Cat# 8146; RRID: AB_10950495 mouse anti-PSD95 synaptic systems Cat# 124011; RRID: AB_10804286 mouse anti-E6AP Sigma-Aldrich Cat# E8655; RRID: AB_261956 rabbit anti-cleaved caspase-3 Cell Signaling Technology Cat# 9661; RRID: AB_2341188 anti-GAPDH EMD Millipore Cat# MAB374; RRID: AB_2107445 mouse anti-a tubulin Sigma-Aldrich Cat# T9026; RRID: AB_477593 rabbit anti-cleaved tubulin (TubDCasp6) provided kindly by Andrea LeBlanc at McGill University N/A mouse anti-NeuN EMD Millipore Cat# MAB377; RRID: AB_2298772 rabbit anti-Ubiquitin Abcam Cat# ab19247; RRID: AB_444805 mouse-IgG-HRP Bio-Rad Cat# 170-6516; RRID: AB_11125547 Rabbit-IgG-HRP Bio-Rad Cat# 170-6515; RRID: AB_11125142 mouse Alexa Fluor 488 Invitrogen Cat# A21121; RRID: AB_2535764 rabbit Alexa Fluor 488 Invitrogen Cat# A11094; RRID: AB_221544 mouse Alexa Fluor 555 Invitrogen Cat# A21127; RRID: AB_141596 rabbit Alexa Fluor 555 Invitrogen Cat# A21428; RRID: AB_2535849

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: Figure 8. 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 mm. (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 mm. (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 G SEM. Error bars represent SEM, *p < 0.05, **p < 0.01.

Article Snippet: Antibodies mouse anti-USP7 Santa Cruz Cat# sc-137008; RRID: AB_2214163 rabbit anti-XIAP Bioss Antibodies Cat# bs-1281r; RRID: AB_10856518 rabbit anti-XIAP Sigma-Aldrich Cat# PRS3331; RRID: AB_1858883 mouse anti-FLAG tag DYKDDDDK Cell Signaling Technology Cat# 8146; RRID: AB_10950495 mouse anti-PSD95 synaptic systems Cat# 124011; RRID: AB_10804286 mouse anti-E6AP Sigma-Aldrich Cat# E8655; RRID: AB_261956 rabbit anti-cleaved caspase-3 Cell Signaling Technology Cat# 9661; RRID: AB_2341188 anti-GAPDH EMD Millipore Cat# MAB374; RRID: AB_2107445 mouse anti-a tubulin Sigma-Aldrich Cat# T9026; RRID: AB_477593 rabbit anti-cleaved tubulin (TubDCasp6) provided kindly by Andrea LeBlanc at McGill University N/A mouse anti-NeuN EMD Millipore Cat# MAB377; RRID: AB_2298772 rabbit anti-Ubiquitin Abcam Cat# ab19247; RRID: AB_444805 mouse-IgG-HRP Bio-Rad Cat# 170-6516; RRID: AB_11125547 Rabbit-IgG-HRP Bio-Rad Cat# 170-6515; RRID: AB_11125142 mouse Alexa Fluor 488 Invitrogen Cat# A21121; RRID: AB_2535764 rabbit Alexa Fluor 488 Invitrogen Cat# A11094; RRID: AB_221544 mouse Alexa Fluor 555 Invitrogen Cat# A21127; RRID: AB_141596 rabbit Alexa Fluor 555 Invitrogen Cat# A21428; RRID: AB_2535849

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: Figure 9. 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 mm; right: Scale bar = 200 mm) (C) can be detected in the whole brain around P30 to P60 ( left: Scale bar = 500 mm; right: Scale bar = 100 mm). (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 mm. 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 G SEM. Error bars represent SEM, *p < 0.05, **p < 0.01.

Article Snippet: Antibodies mouse anti-USP7 Santa Cruz Cat# sc-137008; RRID: AB_2214163 rabbit anti-XIAP Bioss Antibodies Cat# bs-1281r; RRID: AB_10856518 rabbit anti-XIAP Sigma-Aldrich Cat# PRS3331; RRID: AB_1858883 mouse anti-FLAG tag DYKDDDDK Cell Signaling Technology Cat# 8146; RRID: AB_10950495 mouse anti-PSD95 synaptic systems Cat# 124011; RRID: AB_10804286 mouse anti-E6AP Sigma-Aldrich Cat# E8655; RRID: AB_261956 rabbit anti-cleaved caspase-3 Cell Signaling Technology Cat# 9661; RRID: AB_2341188 anti-GAPDH EMD Millipore Cat# MAB374; RRID: AB_2107445 mouse anti-a tubulin Sigma-Aldrich Cat# T9026; RRID: AB_477593 rabbit anti-cleaved tubulin (TubDCasp6) provided kindly by Andrea LeBlanc at McGill University N/A mouse anti-NeuN EMD Millipore Cat# MAB377; RRID: AB_2298772 rabbit anti-Ubiquitin Abcam Cat# ab19247; RRID: AB_444805 mouse-IgG-HRP Bio-Rad Cat# 170-6516; RRID: AB_11125547 Rabbit-IgG-HRP Bio-Rad Cat# 170-6515; RRID: AB_11125142 mouse Alexa Fluor 488 Invitrogen Cat# A21121; RRID: AB_2535764 rabbit Alexa Fluor 488 Invitrogen Cat# A11094; RRID: AB_221544 mouse Alexa Fluor 555 Invitrogen Cat# A21127; RRID: AB_141596 rabbit Alexa Fluor 555 Invitrogen Cat# A21428; RRID: AB_2535849

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: Figure 10. 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.

Article Snippet: Antibodies mouse anti-USP7 Santa Cruz Cat# sc-137008; RRID: AB_2214163 rabbit anti-XIAP Bioss Antibodies Cat# bs-1281r; RRID: AB_10856518 rabbit anti-XIAP Sigma-Aldrich Cat# PRS3331; RRID: AB_1858883 mouse anti-FLAG tag DYKDDDDK Cell Signaling Technology Cat# 8146; RRID: AB_10950495 mouse anti-PSD95 synaptic systems Cat# 124011; RRID: AB_10804286 mouse anti-E6AP Sigma-Aldrich Cat# E8655; RRID: AB_261956 rabbit anti-cleaved caspase-3 Cell Signaling Technology Cat# 9661; RRID: AB_2341188 anti-GAPDH EMD Millipore Cat# MAB374; RRID: AB_2107445 mouse anti-a tubulin Sigma-Aldrich Cat# T9026; RRID: AB_477593 rabbit anti-cleaved tubulin (TubDCasp6) provided kindly by Andrea LeBlanc at McGill University N/A mouse anti-NeuN EMD Millipore Cat# MAB377; RRID: AB_2298772 rabbit anti-Ubiquitin Abcam Cat# ab19247; RRID: AB_444805 mouse-IgG-HRP Bio-Rad Cat# 170-6516; RRID: AB_11125547 Rabbit-IgG-HRP Bio-Rad Cat# 170-6515; RRID: AB_11125142 mouse Alexa Fluor 488 Invitrogen Cat# A21121; RRID: AB_2535764 rabbit Alexa Fluor 488 Invitrogen Cat# A11094; RRID: AB_221544 mouse Alexa Fluor 555 Invitrogen Cat# A21127; RRID: AB_141596 rabbit Alexa Fluor 555 Invitrogen Cat# A21428; RRID: AB_2535849

Techniques: Over Expression, Activity Assay, Infection

Journal: Immunology

Article Title: Small‐molecule inhibitors of ubiquitin‐specific protease 7 enhance type‐I interferon antiviral efficacy by destabilizing SOCS1

doi: 10.1111/imm.13147

Figure Lengend Snippet: Viral infection promotes protein expression of USP7 that up‐regulates SOCS1 protein levels at the late stage of viral infection. (a) Western blot analysis of protein levels of SOCS1 and JOSD1 in A549 cells infected with H1N1 [multiplicity of infection (MOI) = 1·0] for the indicated times. (b) Western blot analysis of exogenously expressed Myc‐SOCS1 levels in HEK293T cells transfected with Myc‐SOCS1 and then infected with vesicular stomatitis virus (VSV) (MOI = 1·0). (c) Immunoprecipitation analysis of polyubiquitination of SOCS1 in HEK293T cells transfected with Myc‐SOCS1 and then infected with VSV (MOI = 1·0). (d) Western blot analysis of Myc‐SOCS1 protein levels in HEK293T cells transfected with Myc‐SOCS1 and different DUBs, followed by treatment with or without cycloheximide (50 μm) for 12 hr. (e) Western blot analysis of Myc‐SOCS1 protein levels in A549 cells co‐transfected with Myc‐SOCS1, control shRNAs (shCON) or shRNAs against human USP7 (shUSP7) as indicated and then infected with H1N1 (MOI = 1·0). (f) Western blot analysis of Myc‐SOCS1 protein levels in A549 cells transfected with Myc‐SOCS1 and then treated with dimethylsulfoxide or P5091 (100 nm) for 12 hr, followed by infection with VSV (MOI = 1·0). (g) Western blot analysis of USP7 protein levels in A549 cells infected with H1N1 (MOI = 1·0) for the indicated times. (h,i) Western blot analysis of USP7 protein levels in HEK293T cells infected with Sendai virus (SeV) (MOI = 1·0) (h) or VSV (MOI = 1·0) (i). The densities of protein bands were quantified with imageJ. (j) Quantitative PCR analysis of USP7 mRNA levels in HEK293T cells infected with VSV (MOI = 1·0), SeV (MOI = 1·0) or H1N1 (MOI = 1·0) for 24 hr. (k) Quantitative PCR analysis of USP7 mRNA levels in HEK293T cells pretreated with pyrrolidine dithiocarbamate ammonium (PDTC) (nuclear factor‐κB inhibitor, 10 μm) for 1 hr and then infected with VSV (MOI = 1·0) for 24 hr. The data were shown as fold change normalized to that in uninfected control cells. (l) Western blot analysis of Myc‐SOCS1 protein levels in A549 cells co‐transfected with Myc‐SOCS1, together with control shRNAs (–) or three shUSP7 as indicated. (m) Western blot analysis of Myc‐SOCS1 protein levels in A549 cells co‐transfected with Myc‐SOCS1 and increasing amounts of Flag‐USP7. (n) Western blot analysis of SOCS1 protein levels in A549 cells co‐transfected with Flag‐USP7 (WT or inactive C223S mutant) as indicated

Article Snippet: USP7 inhibitors (P5091 and {"type":"entrez-protein","attrs":{"text":"P22077","term_id":"134707"}} P22077 ), Trypan Blue and PDTC ammonium (PDTC) were purchased from Selleck Chemicals (Houston, TX).

Techniques: Infection, Expressing, Western Blot, Transfection, Virus, Immunoprecipitation, Control, Real-time Polymerase Chain Reaction, Mutagenesis

Journal: Immunology

Article Title: Small‐molecule inhibitors of ubiquitin‐specific protease 7 enhance type‐I interferon antiviral efficacy by destabilizing SOCS1

doi: 10.1111/imm.13147

Figure Lengend Snippet: USP7 interacts with and stabilizes SOCS1 protein. (a,b) Immunoprecipitation analysis of the interaction between Flag‐USP7 and Myc‐SOCS1 in A549 cells co‐transfected with Myc‐SOCS1 and Flag‐USP7 using anti‐Myc (a) or anti‐Flag (b) antibodies for immunoprecipitation. (c) Immunoprecipitation analysis of the interaction between endogenous USP7 and SOCS1 in A549 cells using either the isotype IgG (−) or anti‐USP7 antibodies (USP7) for immunoprecipitation. (d) Immunoprecipitation analysis of the interaction between endogenous USP7 and SOCS1 in A549 cells using either the isotype IgG (−) or anti‐SOCS1 antibodies (SOCS1) for immunoprecipitation. (e) Western blot analysis of Myc‐SOCS1 protein levels in HEK293T cells transfected with Myc‐SOCS1, together with shCON or shUSP7, followed by treatment with cycloheximide (50 μm) as indicated. (f) Western blot analysis of Myc‐SOCS1 protein levels in HEK293T cells transfected with Myc‐SOCS1, together with shCON (−) or shUSP7, followed by treatment with dimethylsulfoxide or MG132 (10 μm) for 12 hr

Article Snippet: USP7 inhibitors (P5091 and {"type":"entrez-protein","attrs":{"text":"P22077","term_id":"134707"}} P22077 ), Trypan Blue and PDTC ammonium (PDTC) were purchased from Selleck Chemicals (Houston, TX).

Techniques: Immunoprecipitation, Transfection, Western Blot

Journal: Immunology

Article Title: Small‐molecule inhibitors of ubiquitin‐specific protease 7 enhance type‐I interferon antiviral efficacy by destabilizing SOCS1

doi: 10.1111/imm.13147

Figure Lengend Snippet: USP7 removes polyubiquitination of SOCS1 dependently on its deubiquitinase activity. (a) Immunoprecipitation analysis of polyubiquitination of Myc‐SOCS1 in HEK293T cells co‐transfected with Myc‐SOCS1, HA‐ub and Flag‐USP7 as indicated. (b) Immunoprecipitation analysis of polyubiquitination of Myc‐SOCS1 in A549 cells co‐transfected with Myc‐SOCS1 and increasing amounts of Flag‐USP7. (c) Immunoprecipitation analysis of polyubiquitination of endogenous SOCS1 in HEK293T cells transfected with empty vectors (−) or Flag‐USP7. (d) Immunoprecipitation analysis of polyubiquitination of SOCS1 in HEK293T cells co‐transfected with Myc‐SOCS1, HA‐ub and shUSP7 as indicated. (e) Immunoprecipitation analysis of polyubiquitination of SOCS1 in HEK293T cells co‐transfected with Myc‐SOCS1 and Flag‐USP7 (WT or inactive C223S mutant) as indicated

Article Snippet: USP7 inhibitors (P5091 and {"type":"entrez-protein","attrs":{"text":"P22077","term_id":"134707"}} P22077 ), Trypan Blue and PDTC ammonium (PDTC) were purchased from Selleck Chemicals (Houston, TX).

Techniques: Activity Assay, Immunoprecipitation, Transfection, Mutagenesis

Journal: Immunology

Article Title: Small‐molecule inhibitors of ubiquitin‐specific protease 7 enhance type‐I interferon antiviral efficacy by destabilizing SOCS1

doi: 10.1111/imm.13147

Figure Lengend Snippet: USP7 regulates interferon type I (IFN‐I) ‐induced Janus kinase–signal transducer and activator of transcription (JAK–STAT1) activation through SOCS1. (a) Western blot analysis of STAT1 phosphorylation on tyrosine 701 (p‐STAT1) in HEK293T cells transfected with control shRNA (−) or three shUSP7 and then treated with IFN‐α (1000 IU/ml) for 30 min. (b) Western blot analysis of p‐STAT1 in HEK293T cells transfected with shCON or shUSP7 and then treated with IFN‐α (1000 IU/ml) as indicated. (c) Western blot analysis of p‐STAT1 in HEK293T cells transfected with empty vectors (CON) or Flag‐USP7 and then treated with IFN‐α (1000 IU/ml) as indicated. (d) Western blot analysis of p‐STAT1 in HEK293T cells transfected with Flag‐USP7 and then treated with IFN‐α (0, 1000, 3000 IU/ml) for 30 min. (e) Western blot analysis of p‐STAT1 in HEK293T cells transfected with empty vectors (−) or Flag‐USP7 (WT or inactive C223S mutant) and then treated with IFN‐α (1000 IU/ml) as indicated. (f) Western blot analysis of phosphorylated Tyk2 on tyrosine 1054/1055 (p‐Tyk2) and phosphorylated JAK1 on serine 1022/1023 (p‐JAK1) in HEK293T cells transfected with Flag‐USP7 and then treated with IFN‐α (1000 IU/ml) as indicated. (g) Western blot analysis of endogenous SOCS1 protein levels in stable SOCS1‐knockdown HeLa cells (shSOCS1: 1# and 2#) or stable control‐shRNA HeLa cells (shCON). (h) Western blot analysis of p‐STAT1 levels in stable SOCS1‐knockdown HeLa cells transfected with Flag‐USP7 and then treated with IFN‐α (1000 IU/ml) for 30 min

Article Snippet: USP7 inhibitors (P5091 and {"type":"entrez-protein","attrs":{"text":"P22077","term_id":"134707"}} P22077 ), Trypan Blue and PDTC ammonium (PDTC) were purchased from Selleck Chemicals (Houston, TX).

Techniques: Activation Assay, Western Blot, Phospho-proteomics, Transfection, Control, shRNA, Mutagenesis, Knockdown

Journal: Immunology

Article Title: Small‐molecule inhibitors of ubiquitin‐specific protease 7 enhance type‐I interferon antiviral efficacy by destabilizing SOCS1

doi: 10.1111/imm.13147

Figure Lengend Snippet: USP7 regulates the strength of interferon type I (IFN‐I) signaling. (a) The ISRE‐luciferase activity was measured in HEK293T cells transfected with control shRNA (−) or three shUSP7, together with ISRE‐luciferase and Renilla, and then treated with IFN‐α (500 IU/ml) for 18 hr. (b) The ISRE‐luciferase activity was measured in HEK293T cells transfected with Flag‐USP7, together with ISRE‐luciferase and Renilla, and then treated with IFN‐α (500 IU/ml) for 18 hr. (c) The ISRE‐luciferase activity was measured in HEK293T cells transfected with Flag‐USP7 (WT or inactive C223S mutant), together with ISRE‐luciferase and Renilla, and then treated with IFN‐α (200, 500 IU/ml) for 18 hr. (d) Quantitative‐PCR analysis of IFIT1, ISG15 and ISG54 mRNA levels in HEK293T cells transfected with shCON or shUSP7 and then treated with IFN‐α (1000 IU/ml) for 8 hr. (e) Quantitative‐PCR analysis of IFIT1, ISG15 and ISG54 mRNA levels in HEK293T cells transfected with Flag‐USP7 (WT or inactive C223S mutant) and then treated with IFN‐α (1000 IU/ml) for 8 hr. NS, not significant, *P < 0·05, ***P < 0·001 (two‐tailed unpaired Student's t‐test). All data are shown as mean ± SD of three independent replicates

Article Snippet: USP7 inhibitors (P5091 and {"type":"entrez-protein","attrs":{"text":"P22077","term_id":"134707"}} P22077 ), Trypan Blue and PDTC ammonium (PDTC) were purchased from Selleck Chemicals (Houston, TX).

Techniques: Luciferase, Activity Assay, Transfection, Control, shRNA, Mutagenesis, Real-time Polymerase Chain Reaction, Two Tailed Test

Journal: Immunology

Article Title: Small‐molecule inhibitors of ubiquitin‐specific protease 7 enhance type‐I interferon antiviral efficacy by destabilizing SOCS1

doi: 10.1111/imm.13147

Figure Lengend Snippet: USP7 regulates interferon type I (IFN‐I) ‐mediated antiviral activity. (a) Fluorescence microscopy of vesicular stomatitis virus (VSV) with a green fluorescent protein gene (GFP) in HEK293T cells transfected with shCON or shUSP7 and then pretreated with or with IFN‐α (50 IU/ml) for 20 hr, followed by infection with VSV‐GFP [multiplicity of infection (MOI) = 0·5] for 20 hr. Scale bars, 100 µm. (b) Quantitative PCR analysis of mRNA levels of a representative ISG (ISG15) in HEK293T cells transfected with shCON or shUSP7 and then infected with VSV (MOI = 1·0) for 20 hr. (c) Quantitative PCR analysis of VSV viral RNA in HEK293T cells transfected with empty vectors (CON) or Flag‐USP7 and then pretreated with or without IFN‐α (50 IU/ml) for 20 hr, followed by infection with VSV (MOI = 1·0) for 20 hr. (d) Western blot analysis of H1N1‐encoded HA protein levels in HEK293T cells transfected with increasing amounts of Flag‐USP7 and then infected with H1N1 (MOI = 1·0) for 20 hr. (e) Fluorescence microscopy of VSV‐GFP viruses in HEK293T cells transfected with Flag‐USP7 (WT or inactive C223S mutant) and then infected with VSV‐GFP (MOI = 0·5) for 20 hr. Scale bars, 100 µm. ***P < 0·001 (two‐tailed unpaired Student's t‐test). Data are shown as mean ± SD of three independent replicates (b,c)

Article Snippet: USP7 inhibitors (P5091 and {"type":"entrez-protein","attrs":{"text":"P22077","term_id":"134707"}} P22077 ), Trypan Blue and PDTC ammonium (PDTC) were purchased from Selleck Chemicals (Houston, TX).

Techniques: Activity Assay, Fluorescence, Microscopy, Virus, Transfection, Infection, Real-time Polymerase Chain Reaction, Western Blot, Mutagenesis, Two Tailed Test

Journal: Immunology

Article Title: Small‐molecule inhibitors of ubiquitin‐specific protease 7 enhance type‐I interferon antiviral efficacy by destabilizing SOCS1

doi: 10.1111/imm.13147

Figure Lengend Snippet: USP7 Small‐molecule inhibitors enhance interferon type I (IFN‐I) signaling and antiviral efficacy. (a) Immunoprecipitation analysis of polyubiquitination of SOCS1 in HEK293T cells co‐transfected with Myc‐SOCS1 and HA‐ub and then treated with either P5091 (100 nm) or P22077 (100 nm) for 12 hr. (b) Western blot analysis of endogenous SOCS1 protein levels in A549 cells treated with P5091 (100 nm) or P22077 (100 nm) for 12 hr. (c) Western blot analysis of Myc‐SOCS1 protein levels in HEK293T cells transfected with Myc‐SOCS1 and then pretreated with MG132 (10 μm) for 12 hr, followed by treatment with P22077 (100 nm) for 12 hr. (d) Western blot analysis of p‐STAT1 in HEK293T cells pretreated with P22077 or P5091 (100 nm) for 12 hr and then treated with IFN‐α (1000 IU/ml) as indicated. (e) The ISRE‐luciferase activity was measured in HEK293T cells co‐transfected with ISRE‐luciferase and Renilla and then treated with P5091 (100 nm) or P22077 (100 nm) for 12 hr, followed by treatment with IFN‐α (500 IU/ml) for 18 hr. (f) Quantitative PCR analysis of mRNA levels of IFIT1, ISG15 and ISG54 in HEK293T cells pretreated with dimethylsulfoxide or P22077 (100 nm) for 12 hr, and then treated with or without IFN‐α (1000 IU/ml) for 6 hr. (g) Cell viability was measured in HEK293T cells treated with either P5091 or P22077 using different concentrations as indicated. (h) Quantitative PCR analysis of vesicular stomatitis virus (VSV) viral RNA in HEK293T cells pretreated with P5091 or P22077 (20 and 100 nm) for 12 hr and then challenged with VSV [multiplicity of infection (MOI) = 1·0] for 24 hr. (i) Fluorescence microscopy of VSV‐GFP viruses in HEK293T cells pretreated with DMSO, P5091 and P22077 (100 nm) for 12 hr and then treatment with or without IFN‐α (50 IU/ml) for 20 hr, followed by infection with VSV‐GFP (MOI = 0·5) for 24 hr. Scale bars, 100 µm. *P < 0·05, **P < 0·01 and ***P < 0·001 (two‐tailed unpaired Student's t‐test). Data are shown as mean ± SD. of three independent replicates (e,f,h)

Article Snippet: USP7 inhibitors (P5091 and {"type":"entrez-protein","attrs":{"text":"P22077","term_id":"134707"}} P22077 ), Trypan Blue and PDTC ammonium (PDTC) were purchased from Selleck Chemicals (Houston, TX).

Techniques: Immunoprecipitation, Transfection, Western Blot, Luciferase, Activity Assay, Real-time Polymerase Chain Reaction, Virus, Infection, Fluorescence, Microscopy, Two Tailed Test

Journal: Cell Biology and Toxicology

Article Title: Gomisin B promotes RSL3-induced ferroptosis in neuroblastoma by inhibiting the ESR1/USP7/SREBF1 axis

doi: 10.1007/s10565-026-10155-6

Figure Lengend Snippet: USP7 deubiquitinated SREBF1. ( A ) Co-IP was performed to test the binding relationship between USP7 and SREBF1 in SH-SY5Y and IMR-32 cells. ( B ) Localization of USP7 and SREBF1 in SH-SY5Y ans IMR-32 cells detected by immunofluorescence (25 μm). ( C ) Co-IP was adopted to assess SREBF1 expression and ubiquitination level in USP7 knockdown SH-SY5Y cells with MG-132 treatment. ( D ) The SREBF1 expression level was analyzed by western blot in SH-SY5Y cells following 3-MA treatment. ( E ) After knocking down USP7 and treating with CHX, the half-life of SREBF1 in SH-SY5Y cells was detected by western blot. ( F ) HEK293T cells were co-transfected with FLAG‑tagged USP7 (FLAG‑USP7), HA‑tagged SREBF1 (HA‑SREBF1), and Myc‑tagged ubiquitin variants (Myc‑Ub, Myc‑Ub‑K48, or Myc‑Ub‑K63). Cell lysates were immunoprecipitated (IP) with anti‑HA antibody and probed with anti‑Myc antibody to detect ubiquitinated SREBF1. N = 3, ** p < 0.01

Article Snippet: ESR1 (OE-ESR1) and USP7 (OE-USP7) overexpression vectors were acquired from Sango Biotech (Shanghai, China).

Techniques: Co-Immunoprecipitation Assay, Binding Assay, Immunofluorescence, Expressing, Ubiquitin Proteomics, Knockdown, Western Blot, Transfection, Immunoprecipitation

Journal: Cell Biology and Toxicology

Article Title: Gomisin B promotes RSL3-induced ferroptosis in neuroblastoma by inhibiting the ESR1/USP7/SREBF1 axis

doi: 10.1007/s10565-026-10155-6

Figure Lengend Snippet: ESR1 inhibited ferroptosis in NB cells by transcriptionally activating USP7. ( A ) JASPAR ( http://jaspar.genereg.net ) was applied to predict the binding site between ESR1 and USP7 promoter. ( B ) The binding relationship between ESR1 and promoter of USP7 was verified using a dual-luciferase reporter. ( C ) ChIP was utilized to confirm the binding relationship between ESR1 and the upstream promoter region of USP7. SH-SY5Y and IMR-32 cells were transfected with sh-ESR1 or OE-USP7, and RSL3 was used to induce ferroptosis in NB cells. ( D ) RT-qPCR was carried out to measure ESR1 and USP7 levels. ( E ) CCK-8 was implemented to examine cell viability. ( F ) GSH, MDA and LPO levels were tested via commercial kits. ( G ) C-11 BODIPY 581/591 fluorescent probe was deployed to assess lipid ROS level (100 μm). (H) ESR1, USP7, SREBF1 and SCD1 expressions were calculated by western blot. N = 3, * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: ESR1 (OE-ESR1) and USP7 (OE-USP7) overexpression vectors were acquired from Sango Biotech (Shanghai, China).

Techniques: Binding Assay, Luciferase, Transfection, Quantitative RT-PCR, CCK-8 Assay, Western Blot

Journal: Cell Biology and Toxicology

Article Title: Gomisin B promotes RSL3-induced ferroptosis in neuroblastoma by inhibiting the ESR1/USP7/SREBF1 axis

doi: 10.1007/s10565-026-10155-6

Figure Lengend Snippet: Gomisin B promoted ferroptosis in NB cells by regulating the ESR1/USP7/SREBF1 axis. After OE-USP7 or sh-SREBF1 was transfected into the SH-SY5Y and IMR-32 cell lines, NB cells were treated with RSL3 or Gomisin B. The experimental groups were: Control, RSL3, RSL3 + GB, RSL3 + GB + OE-USP7, RSL3 + GB + OE-USP7 + sh-SREBF1. ( A ) USP7 and SREBF1 mRNA expressions were examined by RT-qPCR. ( B ) Cell viability was visualized by CCK-8 assay. ( C ) The commercial kits were administered to analyze GSH, MDA and LPO levels in each group of cells. ( D ) Intracellular lipid ROS level measured by the C-11 BODIPY 581/591 fluorescent probe (100 μm). ( E ) Western blot was adopted to test ESR1, USP7, SREBF1 and SCD1 expressions. N = 3, * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: ESR1 (OE-ESR1) and USP7 (OE-USP7) overexpression vectors were acquired from Sango Biotech (Shanghai, China).

Techniques: Transfection, Control, Quantitative RT-PCR, CCK-8 Assay, Western Blot

Journal: Cell Biology and Toxicology

Article Title: Gomisin B promotes RSL3-induced ferroptosis in neuroblastoma by inhibiting the ESR1/USP7/SREBF1 axis

doi: 10.1007/s10565-026-10155-6

Figure Lengend Snippet: Gomisin B promoted ferroptosis and inhibited tumor growth in NB tumor-bearing nude mice. NB cells were injected subcutaneously into BALB/c nude mice to construct NB tumor-bearing nude mice, and then Gomisin B or RSL3 was injected intraperitoneally every other day. The experimental groups were: Control, DMSO, RSL3, RSL3 + GB. ( A ) Representative pictures of different tumor tissues were taken. ( B ) Tumor growth curves were plotted, and the tumor weight was recorded. ( C ) The commercial kits were utilized to test tumor tissue GSH, MDA and LPO levels. ( D ) C-11 BODIPY 581/591 fluorescent probe was performed to estimate tissue lipid ROS level (50 μm). ( E ) ESR1, USP7, SREBF1 and SCD1 expressions were examined using western blot. N = 5, * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: ESR1 (OE-ESR1) and USP7 (OE-USP7) overexpression vectors were acquired from Sango Biotech (Shanghai, China).

Techniques: Injection, Construct, Control, Western Blot

Journal: Cell Biology and Toxicology

Article Title: Gomisin B promotes RSL3-induced ferroptosis in neuroblastoma by inhibiting the ESR1/USP7/SREBF1 axis

doi: 10.1007/s10565-026-10155-6

Figure Lengend Snippet: Graphical abstract: Gomisin B inhibits the transcriptional activation of USP7 by downregulating ESR1 to suppress the deubiquitination of SREBF1, thereby enhancing NB ferroptosis

Article Snippet: ESR1 (OE-ESR1) and USP7 (OE-USP7) overexpression vectors were acquired from Sango Biotech (Shanghai, China).

Techniques: Activation Assay

Journal: The Journal of Biological Chemistry

Article Title: Ubiquitin-specific Protease 7 Is a Regulator of Ubiquitin-conjugating Enzyme UbE2E1 *

doi: 10.1074/jbc.M113.469262

Figure Lengend Snippet: Analysis of the in vitro interaction between USP7-NTD and UbE2E1. A, schematics of UbE2E1 and USP7 indicating domain organization of the two proteins and the putative USP7 binding motif within the N terminus of UbE2E1. CAT, catalytic domain. B, comparison of the substrate peptide sequences recognized by USP7-NTD, featuring the (P/A)XXS motif, which is also found in the N terminus of UbE2E1. C, GST pulldowns were performed using GST-USP7-NTD and UbE2E1. GST-USP7-NTD fusion protein was incubated with UbE2E1, loaded onto glutathione resin (load (L)), washed with wash buffer (wash (W)), and eluted with Laemmli SDS-PAGE loading dye (eluate (E)). A GST pulldown of UbE2E1 protein with GST alone served as a negative control. GST pulldown experiments also tested the interaction of GST-USP7-NTD and the N-terminal UbE2E1 deletion mutant (ΔN-UbE2E1) and between the GST-USP7-NTD double mutant USP7-NTDDWand UbE2E1.

Article Snippet: HCT116 wild type and USP7 −/− cells were kindly provided by Bert Vogelstein (Johns Hopkins Medical Institutions).

Techniques: In Vitro, Binding Assay, Comparison, Incubation, SDS Page, Negative Control, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Ubiquitin-specific Protease 7 Is a Regulator of Ubiquitin-conjugating Enzyme UbE2E1 *

doi: 10.1074/jbc.M113.469262

Figure Lengend Snippet: Molecular analysis of the USP7-NTD-UbE2E1 interaction A, dissociation constants between USP7-NTD and UbE2E1 peptides were measured by intrinsic tryptophan fluorescence assays. B, the crystal structure of the USP7-NTD·UbE2E1ASTS complex. A ribbon representation of the structure of USP7-NTD (green) with UbE2E1ASTS in stick form (yellow) is shown. The 2Fo − Fc electron density map showing the UbE2E1ASTS peptide is contoured at 1σ. C, the molecular details of the interaction between USP7-NTD and UbE2E1ASTS are shown in stick format with the same color scheme as in B. Hydrogen bonds are indicated by black dashed lines. D, superimposition of UbE2E1ASTS (yellow) with the peptide from viral interferon regulatory factor protein 4, vIRF4ASTS (salmon), showing the similarity in mode of binding within the USP7 peptide binding region.

Article Snippet: HCT116 wild type and USP7 −/− cells were kindly provided by Bert Vogelstein (Johns Hopkins Medical Institutions).

Techniques: Fluorescence, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Ubiquitin-specific Protease 7 Is a Regulator of Ubiquitin-conjugating Enzyme UbE2E1 *

doi: 10.1074/jbc.M113.469262

Figure Lengend Snippet: USP7 and UbE2E1 localization and interaction in vivo. A, immunofluorescence images of U2OS cells are shown after staining for endogenous UbE2E1 (Cy-3) and USP7 (Alexa Fluor 488) proteins. The merged image indicates a partially overlapping localization of USP7 and UbE2E1 in the nucleus. The nuclei were counterstained with DAPI. B, 293T cells transfected with FLAG-UbE2E1 were subject to immunoprecipitation (IP) using mouse IgG (negative control) and anti-FLAG followed by immunoblotting (IB) using antibodies against USP7 and UbE2E1. C, 293T cells transfected with Myc-USP7 were subject to immunoprecipitation using rabbit IgG and anti-Myc followed by immunoblotting against USP7 and UbE2E1 D, endogenous UbE2E1 from 293T cells was immunoprecipitated with a monoclonal antibody against UbE2E1 or mouse IgG followed by immunoblotting with polyclonal rabbit antibodies against USP7 or UbE2E1.

Article Snippet: HCT116 wild type and USP7 −/− cells were kindly provided by Bert Vogelstein (Johns Hopkins Medical Institutions).

Techniques: In Vivo, Immunofluorescence, Staining, Transfection, Immunoprecipitation, Negative Control, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Ubiquitin-specific Protease 7 Is a Regulator of Ubiquitin-conjugating Enzyme UbE2E1 *

doi: 10.1074/jbc.M113.469262

Figure Lengend Snippet: USP7 attenuates UbE2E1-mediated ubiquitination. A, in vitro ubiquitination assays were performed using UbE2E1, UbE2D2, or ΔN-UbE2E1 (as the E2) in the presence of ATP, E1, Ub, the catalytic domain of NEDD4 (as the E3 ligase), and increasing amounts of USP7 (0–2 μg). Total ubiquitinated products (Ub(n))were detected by immunoblotting (IB) using a specific antibody against ubiquitin. Polyubiquitinated products were highlighted in boxes. B, ubiquitin loading assays were carried out in a reaction containing E1, His6-Ub, and ATP with increasing amounts of USP7 (0–2 μg) in the presence of 1 μg of full-length UbE2E1 or ΔN-UbE2E1. The UbE2E2-S∼Ub and ΔN-UbE2E2-S∼Ub intermediates were visualized by immunoblotting, using anti-UbE2E1 (left panel) or anti-His6 epitope tag (right panel). C, in vitro ubiquitination assays were performed as in A with the addition of active USP7 (lanes 3–5) or USP7 pretreated with ubiquitin-aldehyde (lanes 7–9), which blocks the catalytic activity of USP7. NC (negative control) represents a standard ubiquitination reaction in the absence of ATP.

Article Snippet: HCT116 wild type and USP7 −/− cells were kindly provided by Bert Vogelstein (Johns Hopkins Medical Institutions).

Techniques: Ubiquitin Proteomics, In Vitro, Western Blot, Activity Assay, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: Ubiquitin-specific Protease 7 Is a Regulator of Ubiquitin-conjugating Enzyme UbE2E1 *

doi: 10.1074/jbc.M113.469262

Figure Lengend Snippet: USP7 deubiquitinates UbE2E1. A, in vitro ubiquitination assays were performed as in Fig. 4A with increasing amounts of USP7. Total ubiquitination (Ub(n)) was detected using anti-Ub (left panel) and ubiquitinated UbE2E1 was detected using anti-UbE2E1 (right panel). NC (negative control) represents a standard ubiquitination reaction in the absence of ATP. IB, immunoblot. B, UbE2E1 is subject to ubiquitination and accumulated in the presence of MG132 in U2OS cells. Ubiquitinated UbE2E1 was immunoprecipitated (IP) and visualized by immunoblot using anti-ubiquitin. C, ubiquitinated UbE2E1 was subject to USP7 deubiquitination using USP7 or USP7-CS and visualized by immunoblot using anti-ubiquitin.

Article Snippet: HCT116 wild type and USP7 −/− cells were kindly provided by Bert Vogelstein (Johns Hopkins Medical Institutions).

Techniques: In Vitro, Ubiquitin Proteomics, Negative Control, Western Blot, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Ubiquitin-specific Protease 7 Is a Regulator of Ubiquitin-conjugating Enzyme UbE2E1 *

doi: 10.1074/jbc.M113.469262

Figure Lengend Snippet: USP7 stabilizes UbE2E1 in vivo. A, U2OS cells were transfected with nonspecific control siRNA (siControl) or siRNA targeting USP7 (siUSP7) and treated with 10 μg/ml cycloheximide (CHX) for the indicated number of hours or left untreated (lane 0). Cell lysates were subjected to SDS-PAGE and Western blot analysis using antibodies indicated. B, WT HCT116 cells or HCT116 USP7−/− were treated with cycloheximide and harvested for Western blotting as in A. The levels of UbE2E1 were normalized using actin as a loading control and presented as line graphs in the cycloheximide chase experiments. The asterisk represents the statistical analysis comparing levels of UbE2E1 after siRNA treatment with p < 0.01. Error bars indicate S.D. C, WT USP7 and the catalytically inactive mutant C223S, USP7-CS, were transfected into HCT116 USP7−/− cells. Cells were lysed and blotted for UbE2E1 48 h after transfection. The levels of UbE2E1 were normalized using actin as a loading control and presented in a bar graph (bottom). The asterisk represents the statistical analysis comparing levels of UbE2E1 after USP7 rescue with p < 0.01. Error bars indicate S.D.

Article Snippet: HCT116 wild type and USP7 −/− cells were kindly provided by Bert Vogelstein (Johns Hopkins Medical Institutions).

Techniques: In Vivo, Transfection, Control, SDS Page, Western Blot, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Ubiquitin-specific Protease 7 Is a Regulator of Ubiquitin-conjugating Enzyme UbE2E1 *

doi: 10.1074/jbc.M113.469262

Figure Lengend Snippet: USP7 regulates the stability of UbE2E1. USP7 attenuates UbE2E1-mediated total ubiquitination and stabilizes UbE2E1 through an interaction between USP7-NTD and the N-terminal extension of UbE2E1. Inactivation or disruption of the interaction between USP7 and UbE2E1 leads to UbE2E1 destabilization.

Article Snippet: HCT116 wild type and USP7 −/− cells were kindly provided by Bert Vogelstein (Johns Hopkins Medical Institutions).

Techniques: Ubiquitin Proteomics, Disruption

Journal: Cell reports

Article Title: TIFAB Regulates USP15-Mediated p53 Signaling during Stressed and Malignant Hematopoiesis

doi: 10.1016/j.celrep.2020.01.093

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: For non-fluorescent ubiquitin hydrolysis assays, 2.5 ug of USP15 or USP7 (LifeSensors, DB502) was pre-incubated with Mock buffer or with 3.5 ug recombinant TIFAB brought to 25 uL total volume using assay buffer, and incubated for 30 minutes at 37°C.

Techniques: Ubiquitin Proteomics, Virus, Plasmid Preparation, Recombinant, Silver Staining, Transfection, Purification, Lysis, Microarray, Negative Control, Software

Journal: Acta Pharmaceutica Sinica. B

Article Title: Function, mechanism and drug discovery of ubiquitin and ubiquitin-like modification with multiomics profiling for cancer therapy

doi: 10.1016/j.apsb.2023.07.019

Figure Lengend Snippet: List of representative anticancer small molecules by targeting E2s, CRLs or DUBs.

Article Snippet: Additionally, the researchers of Hybrigenics have presented a series of PyrzPPip-derived USP7 inhibitors.

Techniques: In Vitro, Inhibition