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a. Outline of this study. Comparative analysis of <t>USP7</t> inhibition or degradation in PDAC and melanoma will be carried out using customized inhibitors and degraders. b. Chemical structures of previously described USP7 inhibitors as well as of NK192 (chimera derived from potent USP7 inhibitors FT671 and Compound 5) which was used as USP7 ligand for the PROTAC library synthesis. Half-maximal inhibition (IC 50 ) values were derived from data shown in panel c. NK264 represents a Biotin-functionalized variant of NK192, which was used for pulldown experiments shown in panel d. c. Ubiquitin-Rhodamine110Gly (Ub-RhoG) cleavage assay to determine inhibitory potency of compounds shown in b. Full length human USP7 was incubated with the respective compounds, the fluorogenic substrate was added and residual activity was read out through fluorescence measurements. d. Competitive pulldown experiment to assess proteome-wide specificity of NK192. Biotin-functionalized NK264 was immobilized on beads. Proteins enriched from Panc89 lysate either treated with DMSO or with NK192 (10 µM, 500x IC 50 ) were quantified by mass spectrometry.

Usp7 Fragment, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Targeted degradation of USP7 in solid cancer cells reveals disparate effects of deubiquitinase inhibition vs. acute protein depletion"

Article Title: Targeted degradation of USP7 in solid cancer cells reveals disparate effects of deubiquitinase inhibition vs. acute protein depletion

Journal: bioRxiv

doi: 10.1101/2025.07.01.662351

a. Outline of this study. Comparative analysis of USP7 inhibition or degradation in PDAC and melanoma will be carried out using customized inhibitors and degraders. b. Chemical structures of previously described USP7 inhibitors as well as of NK192 (chimera derived from potent USP7 inhibitors FT671 and Compound 5) which was used as USP7 ligand for the PROTAC library synthesis. Half-maximal inhibition (IC 50 ) values were derived from data shown in panel c. NK264 represents a Biotin-functionalized variant of NK192, which was used for pulldown experiments shown in panel d. c. Ubiquitin-Rhodamine110Gly (Ub-RhoG) cleavage assay to determine inhibitory potency of compounds shown in b. Full length human USP7 was incubated with the respective compounds, the fluorogenic substrate was added and residual activity was read out through fluorescence measurements. d. Competitive pulldown experiment to assess proteome-wide specificity of NK192. Biotin-functionalized NK264 was immobilized on beads. Proteins enriched from Panc89 lysate either treated with DMSO or with NK192 (10 µM, 500x IC 50 ) were quantified by mass spectrometry.

Figure Legend Snippet: a. Outline of this study. Comparative analysis of USP7 inhibition or degradation in PDAC and melanoma will be carried out using customized inhibitors and degraders. b. Chemical structures of previously described USP7 inhibitors as well as of NK192 (chimera derived from potent USP7 inhibitors FT671 and Compound 5) which was used as USP7 ligand for the PROTAC library synthesis. Half-maximal inhibition (IC 50 ) values were derived from data shown in panel c. NK264 represents a Biotin-functionalized variant of NK192, which was used for pulldown experiments shown in panel d. c. Ubiquitin-Rhodamine110Gly (Ub-RhoG) cleavage assay to determine inhibitory potency of compounds shown in b. Full length human USP7 was incubated with the respective compounds, the fluorogenic substrate was added and residual activity was read out through fluorescence measurements. d. Competitive pulldown experiment to assess proteome-wide specificity of NK192. Biotin-functionalized NK264 was immobilized on beads. Proteins enriched from Panc89 lysate either treated with DMSO or with NK192 (10 µM, 500x IC 50 ) were quantified by mass spectrometry.

Techniques Used: Inhibition, Derivative Assay, Variant Assay, Ubiquitin Proteomics, Cleavage Assay, Incubation, Activity Assay, Fluorescence, Mass Spectrometry

a. Schematic of the synthesis of USP7-targeting degraders. Functionalized USP7 ligands (top left, pink triangle) were either coupled directly to VHL-linker conjugates (yellow triangle with grey stick, through steps A and B) or first further functionalized with linkers followed by coupling to the VHL ligand (yellow triangle, through steps A, C and D). See the Supporting Information for detailed chemical procedures. b. Chemical structures of USP7-targeting degrader library. A general structure of the PROTACs is shown on the left, consisting of a USP7 binding ligand (top) and E3 ligase binding ligand (bottom) separated by a linker. Linker attachment points to both ligands are indicated by waved lines (black: USP7, red: VHL). Linkers are shown in the table on the right. c.-d. Assessment of USP7 levels upon treatment with compounds in Panc89 cells (c) and Ma-Mel-47 cells (d). Cells were treated with 5 µM of indicated compounds for 24 h, and cell lysates were analyzed through Western blots with indicated antibodies. See the Supporting Information for uncropped blots. e. Chemical structure of NK225.

Figure Legend Snippet: a. Schematic of the synthesis of USP7-targeting degraders. Functionalized USP7 ligands (top left, pink triangle) were either coupled directly to VHL-linker conjugates (yellow triangle with grey stick, through steps A and B) or first further functionalized with linkers followed by coupling to the VHL ligand (yellow triangle, through steps A, C and D). See the Supporting Information for detailed chemical procedures. b. Chemical structures of USP7-targeting degrader library. A general structure of the PROTACs is shown on the left, consisting of a USP7 binding ligand (top) and E3 ligase binding ligand (bottom) separated by a linker. Linker attachment points to both ligands are indicated by waved lines (black: USP7, red: VHL). Linkers are shown in the table on the right. c.-d. Assessment of USP7 levels upon treatment with compounds in Panc89 cells (c) and Ma-Mel-47 cells (d). Cells were treated with 5 µM of indicated compounds for 24 h, and cell lysates were analyzed through Western blots with indicated antibodies. See the Supporting Information for uncropped blots. e. Chemical structure of NK225.

Techniques Used: Binding Assay, Western Blot

a. Chemical structures of improved VHL-targeting PROTACs NK250, NK266 and non-VHL binding control compound NK245. The additional methyl groups in the VHL ligand in NK250 and NK266 (leading to enhanced VHL binding) as well as the inversed hydroxyproline stereocenter in NK245 (abrogating VHL binding) are highlighted. b. USP7 degradation assay. Panc89 cells (left) or Ma-Mel-47 cells (right) were treated with 5 µM of indicated compounds for 24 h and analyzed by Western blot. See the Supporting Information for uncropped blots. c. Assessment of USP7 degradation efficiency. Panc89 cells (left) or Ma-Mel-47 cells (right) were treated with increasing concentrations of either NK250 (left) or NK266 (right). d. Determination of degradation kinetics. Panc89 cells (left) or Ma-Mel47 cells (right) were treated with of 1 µM of NK250 (left) and NK266 (right) for indicated times. e. Degradation rescue experiment. Panc89 cells (left) or Ma-Mel47 cells (right) were pretreated for 2 h with either DMSO, NEDDylation inhibitor MLN4924 (500 nM), proteasome inhibitor Carfilzomib (CFZ, 250 nM) or VHL ligand NK249 (10 µM) followed by 1 µM of NK250 or N266 for 20 h. f. Chemical structure of VHL ligand NK249. g. Orthogonal confirmation of USP7 depletion by immune fluorescence. Panc89 cells were treated with 1 µM NK250 for 24 h before staining for USP7 (green), actin (red), and with DAPI (blue). Scale bar: 10 µm.

Figure Legend Snippet: a. Chemical structures of improved VHL-targeting PROTACs NK250, NK266 and non-VHL binding control compound NK245. The additional methyl groups in the VHL ligand in NK250 and NK266 (leading to enhanced VHL binding) as well as the inversed hydroxyproline stereocenter in NK245 (abrogating VHL binding) are highlighted. b. USP7 degradation assay. Panc89 cells (left) or Ma-Mel-47 cells (right) were treated with 5 µM of indicated compounds for 24 h and analyzed by Western blot. See the Supporting Information for uncropped blots. c. Assessment of USP7 degradation efficiency. Panc89 cells (left) or Ma-Mel-47 cells (right) were treated with increasing concentrations of either NK250 (left) or NK266 (right). d. Determination of degradation kinetics. Panc89 cells (left) or Ma-Mel47 cells (right) were treated with of 1 µM of NK250 (left) and NK266 (right) for indicated times. e. Degradation rescue experiment. Panc89 cells (left) or Ma-Mel47 cells (right) were pretreated for 2 h with either DMSO, NEDDylation inhibitor MLN4924 (500 nM), proteasome inhibitor Carfilzomib (CFZ, 250 nM) or VHL ligand NK249 (10 µM) followed by 1 µM of NK250 or N266 for 20 h. f. Chemical structure of VHL ligand NK249. g. Orthogonal confirmation of USP7 depletion by immune fluorescence. Panc89 cells were treated with 1 µM NK250 for 24 h before staining for USP7 (green), actin (red), and with DAPI (blue). Scale bar: 10 µm.

Techniques Used: Binding Assay, Control, Degradation Assay, Western Blot, Fluorescence, Staining

a. Schematic representation of HiBiT endpoint degradation assay. b. HiBiT-based quantification of USP7 levels. MV4-11 cells stably expressing HiBiT-USP7 generated through lentiviral transduction were treated with PROTACs for either 6 or 24 h. Remaining HiBiT-USP7 could be detected through the luciferase signal using HiBiT Lytic Detection System. Data are shown as mean ± S.D. (N=3), normalized to DMSO treatment. Half-maximal degradation concentrations (DC 50 ) derived from these data are given. c. Schematic representation of a cellular ternary complex formation assay. Halotag-VHL- and NanoLuc-USP7-expressing cells are sequentially treated with Halotag-Fluorophor and PROTAC. Compound-induced proximity is read out by a bioluminescence resonance energy transfer (BRET) signal as shown, demonstrating ternary complex formation. d. Cellular ternary complex formation assay. HEK293T cells overexpressing Halotag-VHL and NanoLuc-USP7 were treated with HaloTag NanoBRET 618 Ligand for 20 h, followed by NK250 or NK266 treatment at indicated concentrations for 2 h prior BRET measurement. The determined half-maximal ternary complex formation concentration (EC 50 ) for NK250 is given. Data are shown as mean ± S.D. (N=4). mBu, milli BRET units.

Figure Legend Snippet: a. Schematic representation of HiBiT endpoint degradation assay. b. HiBiT-based quantification of USP7 levels. MV4-11 cells stably expressing HiBiT-USP7 generated through lentiviral transduction were treated with PROTACs for either 6 or 24 h. Remaining HiBiT-USP7 could be detected through the luciferase signal using HiBiT Lytic Detection System. Data are shown as mean ± S.D. (N=3), normalized to DMSO treatment. Half-maximal degradation concentrations (DC 50 ) derived from these data are given. c. Schematic representation of a cellular ternary complex formation assay. Halotag-VHL- and NanoLuc-USP7-expressing cells are sequentially treated with Halotag-Fluorophor and PROTAC. Compound-induced proximity is read out by a bioluminescence resonance energy transfer (BRET) signal as shown, demonstrating ternary complex formation. d. Cellular ternary complex formation assay. HEK293T cells overexpressing Halotag-VHL and NanoLuc-USP7 were treated with HaloTag NanoBRET 618 Ligand for 20 h, followed by NK250 or NK266 treatment at indicated concentrations for 2 h prior BRET measurement. The determined half-maximal ternary complex formation concentration (EC 50 ) for NK250 is given. Data are shown as mean ± S.D. (N=4). mBu, milli BRET units.

Techniques Used: Degradation Assay, Stable Transfection, Expressing, Generated, Transduction, Luciferase, Derivative Assay, Tube Formation Assay, Bioluminescence Resonance Energy Transfer, Concentration Assay

Proteomic analysis of Panc89 cells treated with PROTAC NK250 (a-c) or inhibitor NK192 (d-f) for indicated time points. Volcano blots report proteins identified by data-independent acquisition mass spectrometry in Panc89 cells treated with NK192 (5 µM) or NK250 (1 µM) for 6, 24 or 72 h. Proteins annotated as members of the non-canonical repressive complex 1.6 are highlighted in orange, chromatin bound E3 ligases are highlighted in black, the most upregulated proteins by USP7 inhibition in blue. g.-l. Proteomic analysis of Ma-Mel-47 cells treated with PROTAC NK266 (g-I, 1 µM) or inhibitor NK192 (j-l, 5 µM) for indicated time points.

Figure Legend Snippet: Proteomic analysis of Panc89 cells treated with PROTAC NK250 (a-c) or inhibitor NK192 (d-f) for indicated time points. Volcano blots report proteins identified by data-independent acquisition mass spectrometry in Panc89 cells treated with NK192 (5 µM) or NK250 (1 µM) for 6, 24 or 72 h. Proteins annotated as members of the non-canonical repressive complex 1.6 are highlighted in orange, chromatin bound E3 ligases are highlighted in black, the most upregulated proteins by USP7 inhibition in blue. g.-l. Proteomic analysis of Ma-Mel-47 cells treated with PROTAC NK266 (g-I, 1 µM) or inhibitor NK192 (j-l, 5 µM) for indicated time points.

Techniques Used: Data-independent acquisition, Mass Spectrometry, Inhibition

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Journal: bioRxiv

Article Title: Targeted degradation of USP7 in solid cancer cells reveals disparate effects of deubiquitinase inhibition vs. acute protein depletion

doi: 10.1101/2025.07.01.662351

Figure Lengend Snippet: a. Outline of this study. Comparative analysis of USP7 inhibition or degradation in PDAC and melanoma will be carried out using customized inhibitors and degraders. b. Chemical structures of previously described USP7 inhibitors as well as of NK192 (chimera derived from potent USP7 inhibitors FT671 and Compound 5) which was used as USP7 ligand for the PROTAC library synthesis. Half-maximal inhibition (IC 50 ) values were derived from data shown in panel c. NK264 represents a Biotin-functionalized variant of NK192, which was used for pulldown experiments shown in panel d. c. Ubiquitin-Rhodamine110Gly (Ub-RhoG) cleavage assay to determine inhibitory potency of compounds shown in b. Full length human USP7 was incubated with the respective compounds, the fluorogenic substrate was added and residual activity was read out through fluorescence measurements. d. Competitive pulldown experiment to assess proteome-wide specificity of NK192. Biotin-functionalized NK264 was immobilized on beads. Proteins enriched from Panc89 lysate either treated with DMSO or with NK192 (10 µM, 500x IC 50 ) were quantified by mass spectrometry.

Article Snippet: HiBiT-USP7 construct was cloned by extraction of USP7 fragment from plasmid pQHA-USP7 WT puroR (Addgene #46753) using appropriate restriction sites.

Techniques: Inhibition, Derivative Assay, Variant Assay, Ubiquitin Proteomics, Cleavage Assay, Incubation, Activity Assay, Fluorescence, Mass Spectrometry

Journal: bioRxiv

Article Title: Targeted degradation of USP7 in solid cancer cells reveals disparate effects of deubiquitinase inhibition vs. acute protein depletion

doi: 10.1101/2025.07.01.662351

Figure Lengend Snippet: a. Schematic of the synthesis of USP7-targeting degraders. Functionalized USP7 ligands (top left, pink triangle) were either coupled directly to VHL-linker conjugates (yellow triangle with grey stick, through steps A and B) or first further functionalized with linkers followed by coupling to the VHL ligand (yellow triangle, through steps A, C and D). See the Supporting Information for detailed chemical procedures. b. Chemical structures of USP7-targeting degrader library. A general structure of the PROTACs is shown on the left, consisting of a USP7 binding ligand (top) and E3 ligase binding ligand (bottom) separated by a linker. Linker attachment points to both ligands are indicated by waved lines (black: USP7, red: VHL). Linkers are shown in the table on the right. c.-d. Assessment of USP7 levels upon treatment with compounds in Panc89 cells (c) and Ma-Mel-47 cells (d). Cells were treated with 5 µM of indicated compounds for 24 h, and cell lysates were analyzed through Western blots with indicated antibodies. See the Supporting Information for uncropped blots. e. Chemical structure of NK225.

Article Snippet: HiBiT-USP7 construct was cloned by extraction of USP7 fragment from plasmid pQHA-USP7 WT puroR (Addgene #46753) using appropriate restriction sites.

Techniques: Binding Assay, Western Blot

Journal: bioRxiv

Article Title: Targeted degradation of USP7 in solid cancer cells reveals disparate effects of deubiquitinase inhibition vs. acute protein depletion

doi: 10.1101/2025.07.01.662351

Figure Lengend Snippet: a. Chemical structures of improved VHL-targeting PROTACs NK250, NK266 and non-VHL binding control compound NK245. The additional methyl groups in the VHL ligand in NK250 and NK266 (leading to enhanced VHL binding) as well as the inversed hydroxyproline stereocenter in NK245 (abrogating VHL binding) are highlighted. b. USP7 degradation assay. Panc89 cells (left) or Ma-Mel-47 cells (right) were treated with 5 µM of indicated compounds for 24 h and analyzed by Western blot. See the Supporting Information for uncropped blots. c. Assessment of USP7 degradation efficiency. Panc89 cells (left) or Ma-Mel-47 cells (right) were treated with increasing concentrations of either NK250 (left) or NK266 (right). d. Determination of degradation kinetics. Panc89 cells (left) or Ma-Mel47 cells (right) were treated with of 1 µM of NK250 (left) and NK266 (right) for indicated times. e. Degradation rescue experiment. Panc89 cells (left) or Ma-Mel47 cells (right) were pretreated for 2 h with either DMSO, NEDDylation inhibitor MLN4924 (500 nM), proteasome inhibitor Carfilzomib (CFZ, 250 nM) or VHL ligand NK249 (10 µM) followed by 1 µM of NK250 or N266 for 20 h. f. Chemical structure of VHL ligand NK249. g. Orthogonal confirmation of USP7 depletion by immune fluorescence. Panc89 cells were treated with 1 µM NK250 for 24 h before staining for USP7 (green), actin (red), and with DAPI (blue). Scale bar: 10 µm.

Article Snippet: HiBiT-USP7 construct was cloned by extraction of USP7 fragment from plasmid pQHA-USP7 WT puroR (Addgene #46753) using appropriate restriction sites.

Techniques: Binding Assay, Control, Degradation Assay, Western Blot, Fluorescence, Staining

Journal: bioRxiv

Article Title: Targeted degradation of USP7 in solid cancer cells reveals disparate effects of deubiquitinase inhibition vs. acute protein depletion

doi: 10.1101/2025.07.01.662351

Figure Lengend Snippet: a. Schematic representation of HiBiT endpoint degradation assay. b. HiBiT-based quantification of USP7 levels. MV4-11 cells stably expressing HiBiT-USP7 generated through lentiviral transduction were treated with PROTACs for either 6 or 24 h. Remaining HiBiT-USP7 could be detected through the luciferase signal using HiBiT Lytic Detection System. Data are shown as mean ± S.D. (N=3), normalized to DMSO treatment. Half-maximal degradation concentrations (DC 50 ) derived from these data are given. c. Schematic representation of a cellular ternary complex formation assay. Halotag-VHL- and NanoLuc-USP7-expressing cells are sequentially treated with Halotag-Fluorophor and PROTAC. Compound-induced proximity is read out by a bioluminescence resonance energy transfer (BRET) signal as shown, demonstrating ternary complex formation. d. Cellular ternary complex formation assay. HEK293T cells overexpressing Halotag-VHL and NanoLuc-USP7 were treated with HaloTag NanoBRET 618 Ligand for 20 h, followed by NK250 or NK266 treatment at indicated concentrations for 2 h prior BRET measurement. The determined half-maximal ternary complex formation concentration (EC 50 ) for NK250 is given. Data are shown as mean ± S.D. (N=4). mBu, milli BRET units.

Article Snippet: HiBiT-USP7 construct was cloned by extraction of USP7 fragment from plasmid pQHA-USP7 WT puroR (Addgene #46753) using appropriate restriction sites.

Techniques: Degradation Assay, Stable Transfection, Expressing, Generated, Transduction, Luciferase, Derivative Assay, Tube Formation Assay, Bioluminescence Resonance Energy Transfer, Concentration Assay

Journal: bioRxiv

Article Title: Targeted degradation of USP7 in solid cancer cells reveals disparate effects of deubiquitinase inhibition vs. acute protein depletion

doi: 10.1101/2025.07.01.662351

Figure Lengend Snippet: Proteomic analysis of Panc89 cells treated with PROTAC NK250 (a-c) or inhibitor NK192 (d-f) for indicated time points. Volcano blots report proteins identified by data-independent acquisition mass spectrometry in Panc89 cells treated with NK192 (5 µM) or NK250 (1 µM) for 6, 24 or 72 h. Proteins annotated as members of the non-canonical repressive complex 1.6 are highlighted in orange, chromatin bound E3 ligases are highlighted in black, the most upregulated proteins by USP7 inhibition in blue. g.-l. Proteomic analysis of Ma-Mel-47 cells treated with PROTAC NK266 (g-I, 1 µM) or inhibitor NK192 (j-l, 5 µM) for indicated time points.

Article Snippet: HiBiT-USP7 construct was cloned by extraction of USP7 fragment from plasmid pQHA-USP7 WT puroR (Addgene #46753) using appropriate restriction sites.

Techniques: Data-independent acquisition, Mass Spectrometry, Inhibition

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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1) Product Images from "Targeted degradation of USP7 in solid cancer cells reveals disparate effects of deubiquitinase inhibition vs. acute protein depletion"

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