Journal: The Journal of Biological Chemistry

Article Title: Ubiquitin-specific protease 7-mediated stabilization of discoidin domain receptor 1 drives progression of TP53-Mutant cancers

doi: 10.1016/j.jbc.2025.110515

Figure Lengend Snippet: Mass-spectrum and DUBome identified USP7 as a stabilizing DUB for DDR1. A , endogenous DDR1 was immunoprecipitated from A549 cells, followed by mass spectrometry analysis. A DUBome screen of NSC632839 identified potential targets, including USP1, USP7, USP11, USP46, and USP47. USP7 was identified as a common target through the intersection of results from both methods. B , co-transfection of potential DUB targets and DDR1 in HEK293 T cells was performed to assess the stabilization effect on DDR1. DUBs were detected using either HA- or FLAG-tag-specific antibodies. C , HEK-293T cells were transfected with DDR1-Flag (0.5 μg) and Flag/HA-USP7 (0.5, 1, and 1.5 μg). DDR1 and HA-USP7 expression were detected by immunoblotting using the indicated antibodies. D , co-Immunoprecipitation (Co-IP) was performed with Myc IgG beads or DDR1 antibody in HEK-293T cells overexpressing DDR1-Flag and Myc-USP7. DDR1 and Myc-USP7 were detected in both IP and cell lysate (INPUT) by immunoblotting. E , reciprocal-co-IP was performed in A549 cells using DDR1 or USP7 antibodies, with IgG as a negative control. DDR1 and USP7 were detected in both IP and cell lysate (INPUT) by immunoblotting. Shown are the representative results of 3 independent experiments.

Article Snippet: The recombinant human-derived USP7 protein (purchased from MedChemExpress) was tested for activity in the Ubiquitin-AMC assay in the presence or absence of inhibitors.

Techniques: Immunoprecipitation, Mass Spectrometry, Cotransfection, FLAG-tag, Transfection, Expressing, Western Blot, Co-Immunoprecipitation Assay, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: Ubiquitin-specific protease 7-mediated stabilization of discoidin domain receptor 1 drives progression of TP53-Mutant cancers

doi: 10.1016/j.jbc.2025.110515

Figure Lengend Snippet: USP7 deubiquitinates and stabilizes DDR1, thereby promoting tumor cell growth in vitro and in vivo . A , HEK-293T cells were transfected with Flag-tagged DDR1, Myc-tagged USP7, and His-tagged ubiquitin, and treated with MG132 (20 μM) for 4 h prior to collection. Ubiquitinated DDR1 was pulled down using His IgG beads for detection. Ubiquitinated DDR1, USP7, and β-Actin were assessed by western blotting with the indicated antibodies ( upper panel ). The levels of ubiquitinated DDR1 were quantified as shown ( lower panel ). Mean ± SD (n = 3); Unpaired t test. B and C , USP7 was knocked down (KD) in A549 cells using USP7-silencing puromycin-resistant shRNA-1 via lentiviral transduction, with a scrambled (SCR) shRNA serving as a non-targeting control. Protein levels of DDR1, USP7, and β-Actin were assessed by western blotting with the indicated antibodies. qPCR was used to measure the mRNA levels of USP7 and DDR1, normalized to GAPDH. Mean ± SD (n = 4); Unpaired t test. D , A549 SCR and USP7-KD cells were treated with NSC632839 at varying concentrations for 16 h. DDR1, USP7, and GAPDH protein levels were detected by western blotting with the indicated antibodies. E , USP7 gene knockdown was performed in A549 cells. DDR1 immunoprecipitation (IP) was carried out, and ubiquitin, DDR1, and β-Actin protein levels were detected in both the IP and cell lysate (INPUT) by western blotting with the indicated antibodies ( left panel ). The levels of ubiquitinated DDR1 were quantified as shown ( right panel ). Mean ± SD (n = 3); Unpaired t test. F , long-term proliferation effects: Cells were generated as described in (E), and the growth curves of A549-shUSP7 cells were compared to those of SCR control cells using the CTG assay. Mean ± SD; Two-way ANOVA. G , short-term cytotoxicity assays: Cells were generated as described in (E), and the immediate effects of NSC632839 on different cell groups were assessed using the CTG assay. H , nude mice were implanted subcutaneously with 5e6 A549 cells (n = 5/group), 12 days later, 20 mg/kg (QD) of NSC632839 were given intraperitoneally. I , mice were treated with vehicle or NSC632839 (20 mg/kg) and sacrificed on day 15 of treatment. Tumor size measurements of xenograft mice after vehicle and NSC632839 treatment. Mean ± SEM; Two-way ANOVA. J , effects of 15 days NSC632839 treatment on growth of xenograft model were determined. Mean ± SEM; Unpaired t test. Tumor growth inhibition (TGI) rates were thus determined for both groups using the formula: TGI=((MTVcontrol-MTVtreated)/MTVcontrol) × 100. Shown are the representative results of 3 independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: The recombinant human-derived USP7 protein (purchased from MedChemExpress) was tested for activity in the Ubiquitin-AMC assay in the presence or absence of inhibitors.

Techniques: In Vitro, In Vivo, Transfection, Ubiquitin Proteomics, Western Blot, shRNA, Transduction, Control, Knockdown, Immunoprecipitation, Generated, CTG Assay, Inhibition

Journal: The Journal of Biological Chemistry

Article Title: Ubiquitin-specific protease 7-mediated stabilization of discoidin domain receptor 1 drives progression of TP53-Mutant cancers

doi: 10.1016/j.jbc.2025.110515

Figure Lengend Snippet: TP53 mutation or depletion promotes DDR1 expression and its association with USP7. A , DDR1 protein levels were quantitatively analyzed in DLBCL cell lines with TP53-WT and TP53-MUT. Mean ± SD; Unpaired t test. B , tissue microarray (TMA): Tumor tissue samples from 28 DLBCL patients were classified into 2 groups based on in situ FISH analysis: TP53-WT (n = 22) and TP53-deletion (n = 6). DDR1 expression was assessed by immunohistochemical staining, with staining intensity (scored from 0 to 3) and positivity rate (scored from 0 to 4) evaluated separately. The composite score, ranging from 0 to 12, was calculated as the product of these 2 scores. Scale bar represents 50 μm. C , statistical analysis corresponding to the data shown in panel (B) is presented in this panel. Mean ± SD; Unpaired t test. D , HEK-293T cells were co-transfected with a DDR1 promoter-driven firefly luciferase reporter containing either wild-type (WT) or mutated (MUT) binding sites, with or without TP53, and incubated for 48 h before harvesting for luciferase activity measurement. Mean ± SD (n = 6); One-way ANOVA. E and F , TP53 was knocked down in A549 cells using puromycin-resistant shTP53-1 and shTP53-2 lentiviral constructs, with scrambled (SCR) shRNA as a non-targeting control. DDR1, TP53, and β-Actin protein levels were evaluated by western blotting with the indicated antibodies. qPCR was used to measure the mRNA levels of DDR1 and TP53, normalized to GAPDH. Mean ± SD (n = 6); One-way ANOVA. G , A549 and Pfeiffer cells were treated with NSC632839 for 16 h at concentrations of 0, 2.5, 5, and 10 μM. DDR1, TP53, MMP2, β-Actin, and GAPDH protein levels were assessed by western blotting using the specified antibodies. H , HEK-293T SCR and TP53-KD cells were transfected with Flag-tagged DDR1 and Myc-tagged USP7. Myc pulldown was performed, and DDR1 and Myc protein levels were detected in both immunoprecipitations (IPs) and cell lysates (INPUT) by western blotting using the indicated antibodies. Shown are the representative results of 3 independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: The recombinant human-derived USP7 protein (purchased from MedChemExpress) was tested for activity in the Ubiquitin-AMC assay in the presence or absence of inhibitors.

Techniques: Mutagenesis, Expressing, Microarray, In Situ, Immunohistochemical staining, Staining, Transfection, Luciferase, Binding Assay, Incubation, Activity Assay, Construct, shRNA, Control, Western Blot

Journal: Nucleic Acids Research

Article Title: FACT subunit Spt16 controls UVSSA recruitment to lesion-stalled RNA Pol II and stimulates TC-NER

doi: 10.1093/nar/gkz055

Figure Lengend Snippet: Accumulation kinetics of TC-NER factors reveal a CSA independent UVSSA recruitment. ( A ) Representative images of live cell imaging analysis of GFP-UVSSA, GFP-CSB or CSA-GFP at the indicated time points following local UV-C laser (266 nm) induced damage (LUD) in a sub-nuclear region (indicated by a white arrow); scale bar: 7.5 μm. ( B ) Relative accumulation of the indicated GFP-tagged TC-NER factors. GFP fluorescence intensity at LUD was quantified over time and normalized to pre-damage intensity set at 100 at t = 0 ( n = 25 cells of two independent experiments, mean ± SEM). The moment of damage induction is indicated with a black arrow. A one-way Anova test was performed and P -values <0.001 (***) are depicted. ( C ) Representative images of GFP-UVSSA accumulation at the indicated time points at LUD (indicated by a white arrow). GFP-UVSSA is expressed to functionally complement UV S S-A cells (TC-NER proficient cells) or GFP-UVSSA expressed in CS-A and CS-B cell lines; scale bar: 7.5 μm. ( D ) Relative accumulation of GFP-UVSSA in the indicated cell lines. GFP fluorescence intensity at LUD of was quantified over time and normalized to pre-damage intensity set at 100 at t = 0 ( n = 30 cells, two independent experiments, mean ± SEM). The moment of damage induction is indicated with a black arrow.

Article Snippet: Membranes were blocked with 5% milk in PBS for 1 h at room temperature and incubated with primary antibodies raised against GFP (Roche, 11814460001), CSA/ERCC8 (Abcam, ab137033), USP7 (Bethyl, A300–033A), Spt16, SSRP1 (Santa Cruz Biotechnology, sc-28734 and sc-74536, respectively), Spt16 (Abcam, Ab56855) or Tubulin (Sigma Aldrich, clone B-5–1-2).

Techniques: Live Cell Imaging, Fluorescence

Journal: Nucleic Acids Research

Article Title: FACT subunit Spt16 controls UVSSA recruitment to lesion-stalled RNA Pol II and stimulates TC-NER

doi: 10.1093/nar/gkz055

Figure Lengend Snippet: CSA interaction and recruitment to DNA damage is mediated by distinct UVSSA domains. ( A ) Schematic overview of the protein domains present in UVSSA and the used UVSSA deletion mutants that either lack the VHS domain (ΔVHS) or the DUF2043 domain (ΔDUF). NLS: nuclear localization signal. ( B ) UV sensitivity of UV S S-A cells (-) and UV S S-A cells complemented with GFP-UVSSA (wt), GFP-UVSSA ΔDUF (ΔDUF) or UVSSA ΔVHS-GFP (ΔVHS) was determined by their colony-forming ability, following irradiation with the indicated UV-C doses. The percentage of surviving colonies is plotted against the UV-C dose. The number of colonies counted at 0 J/m 2 is set as 100% survival. Data represents the experiment conducted in triplicate and error bars represent SEM. ( C ) Whole-cell extracts (WCE) of UV S S-A patient cells stably expressing the indicated constructs were subjected to GFP immunoprecipitation. Western blot analysis of the immunoprecipitated proteins was performed using GFP, CSA or USP7 antibodies. WCE: whole-cell extract, IP: Immunoprecipitate. ( D ) Representative images of live cell imaging analysis of GFP-UVSSA or ΔDUF and ΔVHS mutants following local UV-C laser (266 nm) induced damage (indicated by a white arrow); scale bar: 7.5 μm. Right panel: 4× zoomed image to visualize accumulation at 3 s post-damage induction. ( E ) GFP fluorescence intensity of the indicated constructs at LUD was quantified over time and normalized to pre-damage intensity set at 100 at t = 0 ( n = 30 cells of two independent experiments, mean ± SEM). A one-way Anova test was performed and P -values < 0.001 (***) are depicted. The moment of damage induction is indicated with a black arrow.

Article Snippet: Membranes were blocked with 5% milk in PBS for 1 h at room temperature and incubated with primary antibodies raised against GFP (Roche, 11814460001), CSA/ERCC8 (Abcam, ab137033), USP7 (Bethyl, A300–033A), Spt16, SSRP1 (Santa Cruz Biotechnology, sc-28734 and sc-74536, respectively), Spt16 (Abcam, Ab56855) or Tubulin (Sigma Aldrich, clone B-5–1-2).

Techniques: Irradiation, Stable Transfection, Expressing, Construct, Immunoprecipitation, Western Blot, Live Cell Imaging, Fluorescence

Journal: Nucleic Acids Research

Article Title: FACT subunit Spt16 controls UVSSA recruitment to lesion-stalled RNA Pol II and stimulates TC-NER

doi: 10.1093/nar/gkz055

Figure Lengend Snippet: Quantitative interaction proteomics reveal UVSSA interaction partners and required UVSSA-domains. ( A ) Scatter plot of log 2 SILAC ratios of proteins isolated by GFP-pulldown in UV S S-A cells stably expressing either GFP-UVSSA or GFP (non-specific binding control). The experiment was conducted in duplicate with a label swap. The log 2 SILAC ratios of proteins identified in the forward experiment (GFP-UVSSA versus GFP, H/L, x -axis) are plotted against the log 2 SILAC ratios of proteins identified in the reversed experiment (GFP-UVSSA versus GFP, L/H, y -axis). Proteins were classified as specific UVSSA interactors (marked in blue) when log 2 SILAC ratio >0.6 (indicated by gray dotted line) in both replicates. ( B ) GO-term analysis of the 66 proteins identified as specific interactors of UVSSA. A selection of the top 10 enriched biological process pathways is shown. GC: gene count; FDR: false discovery rate. ( C ) Scatter plot of log 2 SILAC ratios of proteins identified in the GFP-pulldowns of wt UVSSA versus ΔVHS, only proteins that were also identified in the GFP-UVSSA versus GFP proteomics experiment are depicted. The experiment was conducted in duplicate, including a label swap. The log 2 SILAC ratios of proteins identified in the forward experiment (wt versus ΔVHS, H/L, x -axis) are plotted against the log 2 SILAC ratio of proteins identified in the reversed experiment (wt versus ΔVHS, L/H, y -axis). The majority of proteins have similar binding ability to the ΔVHS mutant compared to the wt (log 2 SILAC ratio <0.6, proteins marked in gray). Proteins marked in blue represent proteins whose interaction with UVSSA is decreased in the absence of the VHS domain. ( D ) Scatter plot of log 2 SILAC ratios of proteins identified in the GFP-pulldowns of wt UVSSA versus ΔDUF only proteins that were also identified in the GFP-UVSSA versus GFP proteomics experiment are depicted. The experiment was conducted in duplicate, including a label swap. The log 2 SILAC ratios of proteins identified in the forward experiment (wt versus ΔDUF, H/L, x -axis) are plotted against the log 2 SILAC ratio of proteins identified in the reversed experiment (wt versus ΔDUF, L/H, y -axis). Proteins marked in blue have a reduced interaction with UVSSAΔDUF compared to wt (proteins are marked in blue, log 2 SILAC ratio >0.6, gray dotted line marks the threshold). ( E ) Cross-linked nuclear extracts of UV S S-A patient cell line (TA24), stably expressing the indicated constructs were subjected to GFP immunoprecipitation. Non-complemented UV S S-A patient cell line (-) was used as negative binding control. WCE: whole-cell extract, IP: Immunoprecipitation. Western blot analysis of the co-immunoprecipitated proteins was performed for GFP, Spt16, SSRP1, USP7 and CSA.

Article Snippet: Membranes were blocked with 5% milk in PBS for 1 h at room temperature and incubated with primary antibodies raised against GFP (Roche, 11814460001), CSA/ERCC8 (Abcam, ab137033), USP7 (Bethyl, A300–033A), Spt16, SSRP1 (Santa Cruz Biotechnology, sc-28734 and sc-74536, respectively), Spt16 (Abcam, Ab56855) or Tubulin (Sigma Aldrich, clone B-5–1-2).

Techniques: Multiplex sample analysis, Isolation, Stable Transfection, Expressing, Binding Assay, Control, Selection, Mutagenesis, Construct, Immunoprecipitation, Western Blot

Journal: Nucleic Acids Research

Article Title: FACT subunit Spt16 controls UVSSA recruitment to lesion-stalled RNA Pol II and stimulates TC-NER

doi: 10.1093/nar/gkz055

Figure Lengend Snippet: Spt16 mediates UVSSA accumulation on UV-C induced DNA damage. ( A ) Representative images of live-cell imaging analysis of GFP-UVSSA expressing cells transfected with the indicated siRNAs (CTRL is a non-targeting siRNA), following local UV-C laser (266 nm) induced damage (indicated by a white arrow); scale bar: 7.5 μm. (B) Relative GFP-UVSSA accumulation at sites of LUD in cells transfected with the indicated siRNA. GFP fluorescence intensity at LUD was measured over time using live-cell confocal imaging and normalized to pre-damage intensity set at 100 at t = 0 ( n = 30 cells of two independent, pooled experiments, mean ± SEM). A one-way Anova test was performed and P -values <0.001 (***) are depicted. The moment of damage induction is indicated with a black arrow. ( C ) siRNA transfected cells as used in the live-cell imaging experiments (A and B) were lysed directly after the experiment. Lysates were analyzed by western blot with the indicated antibodies. Tubulin was used as loading control. ( D ) Relative GFP-CSB accumulation in CS-B (CS1AN) cells at sites of LUD in cells transfected with the indicated siRNA. GFP fluorescence intensity at LUD was measured over time using live-cell confocal imaging and normalized to pre-damage intensity set at 100 at t = 0 ( n > 25 cells of two independent experiments, mean ± SEM). The black arrow indicated the moment of damage induction. Representative images are shown in . ( E ) Relative GFP-UVSSA accumulation at sites of LUD in control and NAP1L1 depleted cells. Representative images and knock down efficacy are shown in , respectively. GFP fluorescence intensity at LUD was measured over time using live-cell confocal imaging and normalized to pre-damage intensity set at 100 at t = 0 ( n > 30 cells, two independent experiments, mean ± SEM). The black arrow indicates the moment of damage induction. ( F ) XP186LV patient cells (XP-C; GG-NER-deficient) were transfected with non-targeting control (CTRL) siRNA and siRNA against CSB and Spt16. Cells were irradiated with UV-C (8 J/m 2 ) or mock-treated as indicated, and subsequently labelled for 7 h with EdU. The efficacy of the gap-filling synthesis was assessed by measuring the fluorescently labeled, incorporated EdU into the DNA. Amplified UDS signals were quantified (upper panel) by confocal microscopy measurement of the total nuclear fluorescence (Alexa-Fluor 488 nm, n > 170 cells for each condition, two independent experiments, mean ± SEM) and representative images (lower panel) are shown. A two-tailed t -test was performed and P -values < 0.001 (***) are depicted.

Article Snippet: Membranes were blocked with 5% milk in PBS for 1 h at room temperature and incubated with primary antibodies raised against GFP (Roche, 11814460001), CSA/ERCC8 (Abcam, ab137033), USP7 (Bethyl, A300–033A), Spt16, SSRP1 (Santa Cruz Biotechnology, sc-28734 and sc-74536, respectively), Spt16 (Abcam, Ab56855) or Tubulin (Sigma Aldrich, clone B-5–1-2).

Techniques: Live Cell Imaging, Expressing, Transfection, Fluorescence, Imaging, Western Blot, Control, Knockdown, Irradiation, Labeling, Amplification, Confocal Microscopy, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: FACT subunit Spt16 controls UVSSA recruitment to lesion-stalled RNA Pol II and stimulates TC-NER

doi: 10.1093/nar/gkz055

Figure Lengend Snippet: Spt16 is recruited to DNA damage early during TC-NER and independent of UVSSA. ( A ) Representative images of live-cell analysis of stable GFP-H2A expressing TC-NER proficient (HeLa cells, top panel) or UV S S-A (TA24 cells, lower panel) cells. Left panel, unbleached cells. After photobleaching half of the nucleus, (middle panel), local UV-damage (indicated with a white arrow) was inflicted with an UV-C (266 nm) laser in the bleached half of the nucleus. H2A-GFP exchange was imaged over time; scale bar: 7.5 μm. ( B ) The recovery of fluorescence in damaged and undamaged areas of the photobleached half of the nucleus is quantified in time. GFP-H2A exchange rate at LUD is depicted, normalized to the undamaged area ( n = 15 cells from two independent mean ± SEM). ( C ) Relative Spt16-GFP accumulation at sites of LUD in UV S S-A deficient and complemented cells. GFP fluorescence intensity at LUD was measured over time using live-cell confocal imaging and was normalized to pre-damage intensity at t = 0, which was set to 100 ( n = 30 cells, two independent experiments, mean ± SEM). The moment of damage induction is indicated with a black arrow. ( D ) Representative images of live-cell imaging analysis of Spt16-GFP expressed in either UV S S-A deficient or in UV S SA complemented (UVSSA-Flag) cells (lower panel). The white arrow indicates areas of UV-C laser (266 nm) induced DNA damage; scale bar: 7.5 μm. ( E ) Quantification of the GFP fluorescence intensity of cells stably expressing the indicated GFP-tagged NER proteins, GFP-tagged Spt16 or SSRP1. Cells subjected to local UV-C laser (266 nm) induced DNA damage were imaged over time. All cells were imaged and damaged under the exact same conditions. Fluorescence at LUD was normalized to pre-damage fluorescence at t = 0, which was set to 100 ( n = 20 cells, two independent experiments, mean ± SEM). The moment of damage induction is indicated with a black arrow. Right graph shows zoomed graph of the indicated box, to clearly illustrate the kinetics of TC-NER factors. Representative images are shown in .

Article Snippet: Membranes were blocked with 5% milk in PBS for 1 h at room temperature and incubated with primary antibodies raised against GFP (Roche, 11814460001), CSA/ERCC8 (Abcam, ab137033), USP7 (Bethyl, A300–033A), Spt16, SSRP1 (Santa Cruz Biotechnology, sc-28734 and sc-74536, respectively), Spt16 (Abcam, Ab56855) or Tubulin (Sigma Aldrich, clone B-5–1-2).

Techniques: Cell Analysis, Expressing, Fluorescence, Imaging, Live Cell Imaging, Stable Transfection

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Knockdown Proteomics Reveals USP7 as a Regulator of Cell-Cell Adhesion in Colorectal Cancer via AJUBA

doi: 10.1016/j.mcpro.2024.100878

Figure Lengend Snippet: List of siRNA Oligos used for transfection

Article Snippet: USP7 (For COIP) , Santa Cruz Biotechnology , (H-12) sc-137008 , Mouse , 1:1000.

Techniques:

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Knockdown Proteomics Reveals USP7 as a Regulator of Cell-Cell Adhesion in Colorectal Cancer via AJUBA

doi: 10.1016/j.mcpro.2024.100878

Figure Lengend Snippet: Details of primary and secondary antibodies used in Western blot

Article Snippet: USP7 (For COIP) , Santa Cruz Biotechnology , (H-12) sc-137008 , Mouse , 1:1000.

Techniques: Western Blot

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Knockdown Proteomics Reveals USP7 as a Regulator of Cell-Cell Adhesion in Colorectal Cancer via AJUBA

doi: 10.1016/j.mcpro.2024.100878

Figure Lengend Snippet: LS180 is an effective model for investigating USP7 function in colorectal cancer. A , scatterplot comparing the distribution of USP7 gene effect processed scores (CERES, DEMETER2) between the RNAi dataset (y) and CRISPR dataset (x). Selected cell-lines with the largest USP7 gene-effect scores from RNAi and CRISPR are labeled in addition to DLD1, an additional CRC cell-line used in this study. All analysis performed in DepMap portal ( depmap.org ). B , efficient degradation of USP7 at the indicated time points. LS88 colon carcinoma cells stably carrying a pTER-USP7 shRNA construct and a Tet repressor (TR)-construct were treated with 1 μg/ml doxycycline for 24, 48, and 72 h and examined by Western blotting for reduction of endogenous USP7; GAPDH was used as a loading control. C , LS88 cells (with inducible siRNA targeting USP7) were treated with 1 μg/ml concentrations of doxycycline for 72 h before being seeded in 6-well plates and incubated for 7 to 14 days. Graph represents mean ± SD (two tailed, unpaired t test), p ≤ 0.0001.

Article Snippet: USP7 (For COIP) , Santa Cruz Biotechnology , (H-12) sc-137008 , Mouse , 1:1000.

Techniques: CRISPR, Labeling, Stable Transfection, shRNA, Construct, Western Blot, Control, Incubation, Two Tailed Test

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Knockdown Proteomics Reveals USP7 as a Regulator of Cell-Cell Adhesion in Colorectal Cancer via AJUBA

doi: 10.1016/j.mcpro.2024.100878

Figure Lengend Snippet: Proteomic analysis of LS88 with/without doxycycline treatment for USP7 knockdown. A , principal component analysis scores plot showing the clustering of proteins according to their distributions in different groups. Control LS88 represents cells at hour zero (0 h), LS88 treated with doxycycline for 72 h (72 h + doxycycline) or left without treatment for 72 h (72 h). B , heatmap of normalized intensity values (light/low – dark/high) for each group (0 h untreated; 72 h untreated; 72 h + doxycycline). All proteins (N = 444) with p < 0.05 for treated versus untreated (at 72 h) are included. C , volcano plot showing differentially responsive proteins in comparison of 72 h untreated versus 72 h doxycycline-treated cells. Selected proteins from enriched pathway and process categories are labeled. D , annotation analysis of the sets of proteins ( p < 0.05) decreased following doxycycline treatment (after 72 h). Pathways from DAVID tool are sorted by p value and the top 15 most significantly enriched pathways shown. Ubiquitin-mediated proteolysis category is indicated by arrow. E , the top 15 most significantly decreased proteins ( p < 0.05) following doxycycline treatment (after 72 h) that are involved in protein polyubiquitination. TRIM27, a USP7 protein-complex partner, is the most significantly altered protein following USP7 knockdown in this category (Analysis performed in Metascape). Proteins that are known to be direct substrates or interaction partners of USP7 (according to STRING) are indicated with arrow. F , dendrogram showing significant Gene Ontology cellular components for the set of proteins ( p < 0.05) decreased following doxycycline treatment. Enrichment p -values are indicated and only GO terms with FDR p -value <0.05 were included. GO terms associated with cell-cell junction functions are highlighted with orange bar. Dendrogram circle shape size indicates p -value.

Article Snippet: USP7 (For COIP) , Santa Cruz Biotechnology , (H-12) sc-137008 , Mouse , 1:1000.

Techniques: Knockdown, Control, Comparison, Labeling, Ubiquitin Proteomics

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Knockdown Proteomics Reveals USP7 as a Regulator of Cell-Cell Adhesion in Colorectal Cancer via AJUBA

doi: 10.1016/j.mcpro.2024.100878

Figure Lengend Snippet: Western blot analysis of Ajuba abundance following USP7 knockdown. A , total cell lysate treated with doxycycline in different time point were collected and analysis using immunoblotting. B , LS88 cells treated or untreated with doxycycline were examined on Ajuba protein expression. Unpaired Student’s t test was used for three independent experiments. p < 0.0001. GAPDH was used as a loading control. C , total cell lysates from LS88 and (D) HCT116 treated with FT671 (10 μM) for indicated times. GAPDH is a loading control.

Article Snippet: USP7 (For COIP) , Santa Cruz Biotechnology , (H-12) sc-137008 , Mouse , 1:1000.

Techniques: Western Blot, Knockdown, Expressing, Control

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Knockdown Proteomics Reveals USP7 as a Regulator of Cell-Cell Adhesion in Colorectal Cancer via AJUBA

doi: 10.1016/j.mcpro.2024.100878

Figure Lengend Snippet: Immunofluorescence analysis of Ajuba levels following USP7 knockdown in LS88 cells. A , immunofluorescence staining of Ajuba, (B) USP7 ( green ) in LS88 cells treated with doxycycline (DOX+) or untreated (DOX−). Cells were fixed and stained with DAPI ( blue ) to stain nuclei. Scale bars represents 30 μm. C , data were corrected by subtracting local background values from cytoplasm and nucleus mean gray values. Statistical differences were determined by Unpaired t test of percentage change of Ajuba and USP7 in cytoplasm and nuclei in LS88 cells. Data are mean ± SD.

Article Snippet: USP7 (For COIP) , Santa Cruz Biotechnology , (H-12) sc-137008 , Mouse , 1:1000.

Techniques: Immunofluorescence, Knockdown, Staining

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Knockdown Proteomics Reveals USP7 as a Regulator of Cell-Cell Adhesion in Colorectal Cancer via AJUBA

doi: 10.1016/j.mcpro.2024.100878

Figure Lengend Snippet: USP7 interacts with Ajuba and Ajuba knockdown does not affect USP7 protein levels in CRC cells. A , mRNA levels of USP7 in doxycycline-treated (DOX+) or untreated (DOX-) LS88 cells. ∗∗ p < 0.01. Data are mean ± SD. B , mRNA levels of Ajuba in DOX + or DOX- LS88 cells. Unpaired t-tests were used for analysis in order to assess any differences between the means. Experiments were performed in triplicate, n = 3. C , LS88 cells total lysates were immunoprecipitated with anti USP7 antibody or IgG, and Ajuba was analyzed using indicated antibodies in LS88 cells. D , LS88 and LS174T cells expressing Ajuba, control siRNA, or Ajuba siRNA were analyzed by Western blotting and USP7 protein expression were examined using the indicated antibodies. As a loading control, GAPDH protein levels were assessed.

Article Snippet: USP7 (For COIP) , Santa Cruz Biotechnology , (H-12) sc-137008 , Mouse , 1:1000.

Techniques: Knockdown, Immunoprecipitation, Expressing, Control, Western Blot

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Knockdown Proteomics Reveals USP7 as a Regulator of Cell-Cell Adhesion in Colorectal Cancer via AJUBA

doi: 10.1016/j.mcpro.2024.100878

Figure Lengend Snippet: USP7 knockdown and effects on cadherin proteins and their substrates. A , protein expression of N-cadherin and E-cadherin in LS88 cells treated with doxycycline (DOX) for the indicated time. GAPDH was used as a loading control. Adding doxycycline induces USP7 expression in LS88 cells. B , protein expression of N-cadherin and E-cadherin in HCT116 cells transfected with siUSP7 or siControl or left untreated. GAPDH was used as a loading control. C , α-catenin and γ-catenin expression were analyzed by Western blot. N = 3. GAPDH was used as a loading control. D , cell lysates from LS88 cells were immunoprecipitated with USP7; IgG antibodies followed by immunoblotting to examine the impact of USP7 knockdown on E-cadherin and γ-catenin (JUP).

Article Snippet: USP7 (For COIP) , Santa Cruz Biotechnology , (H-12) sc-137008 , Mouse , 1:1000.

Techniques: Knockdown, Expressing, Control, Transfection, Western Blot, Immunoprecipitation

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Knockdown Proteomics Reveals USP7 as a Regulator of Cell-Cell Adhesion in Colorectal Cancer via AJUBA

doi: 10.1016/j.mcpro.2024.100878

Figure Lengend Snippet: USP7 knockdown and effects on desmosome and hemidesmosome protein levels. A , protein expression of ITGA6, (B) ITGB4, and (C) DSG2 were analyzed using Western blot. GAPDH was used as a loading control. D , LS88 cells were transfected with siControl and siAJUBA or left untreated; the cell lysates were subjected to Western blotting with indicated antibodies and graph (E) showing protein levels of JUP in the three conditions. The ordinary one-way-ANOVA was used from three independent experiments. The significant difference between the two groups untreated and siAjuba groups is ∗ p < 0.05. Mean ± SD. F , Ajuba protein was knocked down in LS174T using siRNA and Western blot was performed using AJUBA and JUP antibodies. G and H , cell lysates from LS88 cell were subjected to immunoblotting with AJUBA and α -catenin and N-cadherin antibodies. GAPDH was used as a loading control.

Article Snippet: USP7 (For COIP) , Santa Cruz Biotechnology , (H-12) sc-137008 , Mouse , 1:1000.

Techniques: Knockdown, Expressing, Western Blot, Control, Transfection

Journal: Cell Stress & Chaperones

Article Title: Monocyte USP7-p65 axis mediates immune responses to the immunogenicity of nucleus pulposus

doi: 10.1016/j.cstres.2025.100114

Figure Lengend Snippet: Single-cell RNA sequencing reveals significantly elevated USP7 expression in monocytes. (a) UMAP visualization of single-cell transcriptomic profiles. Each dot represents an individual cell, color-coded according to unsupervised clustering. A total of 14 distinct cell subpopulations (Clusters 0-13) were identified, reflecting the cellular heterogeneity within degenerated disc tissue. (b) Violin plots displaying the expression patterns of the top five marker genes for each cluster, facilitating cell type annotation based on transcriptional signatures. (c) Violin plots showing the expression of canonical monocyte/macrophage marker genes across all clusters. (d) Heatmap depicting the top 150 highly expressed genes in monocyte/macrophage populations derived from mildly, moderately, and severely degenerated tissues, highlighting changes in gene expression associated with disease severity. (e)-(h) KEGG and GO enrichment analyses of highly expressed genes in monocyte/macrophage clusters. KEGG pathways enriched in immune and inflammatory responses, including NOD-like receptor signaling, PPAR signaling, chemokine signaling, and necroptosis (e). GO enrichment results categorized into biological processes, cellular components, and molecular functions (f-h).

Article Snippet: The following antibodies were used in this study: CD11b polyclonal antibody (Abcam, Cat# ab133357); TLR4 polyclonal antibody (Thermo Scientific Cat# PA5-23124); USP7 monoclonal antibody (Proteintech, Cat# 66514-1-Ig); CD86 polyclonal antibody (Proteintech, Cat# 30691-1-AP); HMGB1 polyclonal antibody (Proteintech, Cat# 10829-1-AP); and GAPDH monoclonal antibody (Proteintech, Cat# 60004-1-Ig).

Techniques: RNA Sequencing, Expressing, Marker, Derivative Assay, Gene Expression

Journal: Cell Stress & Chaperones

Article Title: Monocyte USP7-p65 axis mediates immune responses to the immunogenicity of nucleus pulposus

doi: 10.1016/j.cstres.2025.100114

Figure Lengend Snippet: NP cells activate monocytes, leading to stress responses in NP cells. (a) Schematic illustration of the Transwell co-culture system used to assess interactions between NP cells and monocytes (Mos). Three culture conditions were established in the Transwell system: NP–Mos co-culture (referred to as Mos NP or NP Mos , depending on the cell type being analyzed), Mos monoculture ( Mos mono ), and NP monoculture ( NP mono ). (b) qPCR analysis of USP7, TLR4, and HMGB1 mRNA expression in monocytes stimulated with or without NP cells. Data were analyzed using an independent samples t test. ( n = 3). (c) and (d) Western blot analysis of USP7, p65, TLR4, and HMGB1 protein levels in monocytes cultured with or without NP cells for 10 or 24 h (c), with densitometric quantification shown in panel D. Data are presented as mean ± SD and were analyzed using two-way ANOVA ( n = 3 or 4). (e) ELISA quantification of HMGB1, IL-1β, IL-6, and TNF-α levels in the culture medium under monoculture and co-culture conditions at different time points. Data were analyzed using two-way ANOVA ( n = 3). (f) Immunofluorescence analysis of p65 nuclear localization and expression in monocytes under monoculture or co-culture conditions. Quantification is shown on the right. Data were analyzed using two-way ANOVA ( n = 3). (g) CCK-8 assay assessing NP cell viability under monoculture and co-culture conditions at 0, 4, 8, 12, 24, and 48 hours. Statistical analysis was performed using two-way ANOVA ( n = 5). (h) qPCR analysis of IL-1β, IL-6, COX-2, and SOD2 mRNA expression in NP cells cultured alone or co-cultured with monocytes. Data were analyzed using an independent samples t test ( n = 3). (i) DCFH-DA fluorescence assay for ROS accumulation in NP cells under monoculture or co-culture conditions. Statistical significance was assessed using an independent samples t test. Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001. n = 3.

Article Snippet: The following antibodies were used in this study: CD11b polyclonal antibody (Abcam, Cat# ab133357); TLR4 polyclonal antibody (Thermo Scientific Cat# PA5-23124); USP7 monoclonal antibody (Proteintech, Cat# 66514-1-Ig); CD86 polyclonal antibody (Proteintech, Cat# 30691-1-AP); HMGB1 polyclonal antibody (Proteintech, Cat# 10829-1-AP); and GAPDH monoclonal antibody (Proteintech, Cat# 60004-1-Ig).

Techniques: Co-Culture Assay, Expressing, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay, Immunofluorescence, CCK-8 Assay, Fluorescence

Journal: Cell Stress & Chaperones

Article Title: Monocyte USP7-p65 axis mediates immune responses to the immunogenicity of nucleus pulposus

doi: 10.1016/j.cstres.2025.100114

Figure Lengend Snippet: The USP7–p65 axis in monocytes is directly associated with NP cell stress. (a) Monocytes were pretreated with a transduction enhancer for 30 min, followed by standard siRNA transfection targeting USP7 or p65. Knockdown efficiency was validated by qPCR ( n = 3). (b) qPCR analysis of TNF-α, HMGB1, IL-1β, COX-2, and SOD2 mRNA levels in NP cells after 12-hour co-culture with monocytes transfected with control siRNA (si-ctrl), si-USP7, or si-p65 ( n = 3). (C) Intracellular ROS levels in NP cells were assessed using the DCFH-DA probe following 12-hour co-culture with monocytes transfected with si-ctrl, si-USP7, or si-p65 ( n = 3). (d) Immunofluorescence analysis of USP7 expression and subcellular localization in monocytes under monoculture or co-culture conditions. Scale bar: 40 µm. Quantification of mean USP7 fluorescence intensity and nuclear-to-cytoplasmic (N/C) ratio is shown below. (e) Immunofluorescence analysis of p65 expression and localization in monocytes transfected with si-ctrl or si-USP7 under co-culture conditions. Scale bar: 40 µm. Quantitative analysis of mean p65 intensity and N/C ratio is provided. (f) Immunofluorescence analysis of p65 expression and localization in monocytes pretreated with DMSO (0.05%) or the USP7 inhibitor P5091 (5 μM) for 3 h prior to co-culture. Scale bar: 40 µm. Quantitative analysis of mean p65 intensity and N/C ratio is shown. Data are presented as mean ± SD. Statistical significance: * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The following antibodies were used in this study: CD11b polyclonal antibody (Abcam, Cat# ab133357); TLR4 polyclonal antibody (Thermo Scientific Cat# PA5-23124); USP7 monoclonal antibody (Proteintech, Cat# 66514-1-Ig); CD86 polyclonal antibody (Proteintech, Cat# 30691-1-AP); HMGB1 polyclonal antibody (Proteintech, Cat# 10829-1-AP); and GAPDH monoclonal antibody (Proteintech, Cat# 60004-1-Ig).

Techniques: Transduction, Transfection, Knockdown, Co-Culture Assay, Control, Immunofluorescence, Expressing, Fluorescence

Journal: Cell Stress & Chaperones

Article Title: Monocyte USP7-p65 axis mediates immune responses to the immunogenicity of nucleus pulposus

doi: 10.1016/j.cstres.2025.100114

Figure Lengend Snippet: Monocytes promote TNF, IL-1β, and HMGB1 promoter activity via USP7 in response to NP immunogenicity . (a) The ∼1 kb promoter regions of human TNF-α, IL-1β, and HMGB1 were cloned upstream of the firefly luciferase gene in the psiCHECK™−2 dual-luciferase reporter vector, with Renilla luciferase serving as an internal control. Reporter constructs were transfected into THP-1 monocytes for 24 h, followed by three conditions: (i) unstimulated monocytes (Ctrl); (ii) co-culture with NP cells for 12 h (Mos NP-12h ); and (iii) USP7 knockdown monocytes co-cultured with NP cells for 12 h (siUSP7 Mos NP-12h ). Luciferase activity was quantified using the Dual-Luciferase® Reporter Assay System (Promega). Firefly signals were normalized to Renilla, and promoter activity was expressed relative to the Ctrl group. (b) ChIP-qPCR was performed to assess p65 enrichment at the promoter regions of TNF-α, HMGB1, and IL-1β in monocytes under NP stimulation. THP-1 cells were pretreated with a transfection enhancer and divided into three experimental groups: (1) co-cultured with NP cells for 12 h (Mos NP-12h ); (2) transfected with USP7 siRNA prior to co-culture (si-USP7 Mos NP-12h ); and (3) pretreated with the USP7 inhibitor P5091 (5 μM, 3 h) before co-culture (P5091 Mos NP-12h ). Chromatin was immunoprecipitated using an anti-p65 antibody, and promoter-specific DNA enrichment was quantified by qPCR. Results were expressed as a percentage of input (% input). (c) Schematic illustration of the proposed mechanism by which USP7 mediates monocyte activation in response to the immunogenicity of NP cells. Upon stimulation by NP-derived immunogenic factors, monocytes exhibit upregulation and nuclear translocation of USP7, which enhances the transcriptional activity of p65 through a deubiquitination-dependent mechanism. This process leads to activation of the NF-κB signaling pathway, resulting in increased expression of TNF, IL-1β, and HMGB1 in monocytes. These pro-inflammatory mediators subsequently induce inflammatory and oxidative stress responses in NP cells, as evidenced by elevated expression of TNF, IL-1β, and HMGB1, along with decreased expression of the antioxidant enzyme SOD2. This establishes a feed-forward loop of inflammation and oxidative stress between monocytes and NP cells. Data are presented as mean ± standard deviation (SD), n = 3. Statistical significance was assessed by two-way ANOVA: * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The following antibodies were used in this study: CD11b polyclonal antibody (Abcam, Cat# ab133357); TLR4 polyclonal antibody (Thermo Scientific Cat# PA5-23124); USP7 monoclonal antibody (Proteintech, Cat# 66514-1-Ig); CD86 polyclonal antibody (Proteintech, Cat# 30691-1-AP); HMGB1 polyclonal antibody (Proteintech, Cat# 10829-1-AP); and GAPDH monoclonal antibody (Proteintech, Cat# 60004-1-Ig).

Techniques: Activity Assay, Immunopeptidomics, Clone Assay, Luciferase, Plasmid Preparation, Control, Construct, Transfection, Co-Culture Assay, Knockdown, Cell Culture, Reporter Assay, ChIP-qPCR, Immunoprecipitation, Activation Assay, Derivative Assay, Translocation Assay, Expressing, Standard Deviation

Journal: Nature communications

Article Title: USP7/Maged1-mediated H2A monoubiquitination in the paraventricular thalamus: an epigenetic mechanism involved in cocaine use disorder.

doi: 10.1038/s41467-023-44120-2

Figure Lengend Snippet: Fig. 3 | Maged1 inactivation modifies gene expression regulated by the Poly- comb pathway through the USP7-H2A interaction. a Mouse Thalamic RNA Sequencing and Immunoprecipitation (IP) Analysis. b Venn diagram showing dif- ferentially regulated genes in control mice, Mage1loxP mice and Maged1-cKO, Vglut2Cre::Maged1loxP mice (log2|FC | ≥0.5, FDR < 0.05). Proportions of overlapping genes between the control and Maged1-cKO group are indicated (chi-square test, two-sided, **P = 0.0016). c Alluvial plot depicting gene expression changes in the control and their potential modifications in Maged1-cKO mice (log2|FC | ≥0.5, FDR < 0.05). d Curated gene set analysis showing overlapping between literature- based gene categories and unmodified genes in Maged1-cKO mice (top) and scheme of the Polycomb repressive complexes, PRC1 and PRC2 (bottom). IgG or anti-Maged1 antibody-based thalamic IP followed by western blotting (WB) (e) or IP-Mass spectrometry (MS) (f) for Maged1; WT band is indicated with a gray arrow, truncated protein (functional KO) is indicated with a blue arrow. f Mass spectro- metry (MS) results for protein abundance between control mice treated with saline (n = 5) and control mice treated with cocaine (n = 7) (left side), and between control

Article Snippet: USP7 inhibition and in vivo cannula infusion USP7 inhibitor: P5091 (Selleck Chemicals, S7132) was prepared freshly before injection.

Techniques: Gene Expression, RNA Sequencing, Immunoprecipitation, Control, Western Blot, Mass Spectrometry, Functional Assay, Quantitative Proteomics, Saline

Journal: Nature communications

Article Title: USP7/Maged1-mediated H2A monoubiquitination in the paraventricular thalamus: an epigenetic mechanism involved in cocaine use disorder.

doi: 10.1038/s41467-023-44120-2

Figure Lengend Snippet: Fig. 4 | Chronic cocaine administration increases H2A ubiquitination, which is impaired by Maged1 inactivation or UPS7 inhibition. a Method: cocaine administration (intraperitoneal (i.p.), 15 mg/kg) and nuclease-based protein extraction. b Representative western blot (thalamus). c H2A monoubiquitination (ratio between H2Aub and H2A signal intensities) in the NAc (c), PFC (d), and thalamus (e). In panel (c): control saline, n = 6; control cocaine, n = 5; cKO saline, n = 5; cKO cocaine, n = 4, (two-way ANOVA, control versus Maged1 cKO, P = 0.3839; saline versus cocaine, *P = 0.0144; interaction factor, P = 0. 6853). In panel (d): control saline, n = 9; control cocaine, n = 11; cKO saline, n = 10; cKO cocaine, n = 8, (two-way ANOVA, control versus Maged1 cKO, P = 0.8751, saline versus cocaine, **P = 0.0023; interaction factor, P = 0. 8428). In panel (e): control saline, n = 14; control cocaine, n = 20; cKO saline, n = 13; cKO cocaine, n = 18, (two-way ANOVA, control versus Maged1-cKO, P = 0.0849; saline versus cocaine, P = 0.4907; inter- action factor, ¶P = 0.0246, Sidak’s test (saline versus cocaine), control *P = 0.0281). f Cocaine sensitization scheme during USP7 inhibition (P5091, i.p., 10 mg/kg;

Article Snippet: USP7 inhibition and in vivo cannula infusion USP7 inhibitor: P5091 (Selleck Chemicals, S7132) was prepared freshly before injection.

Techniques: Ubiquitin Proteomics, Inhibition, Protein Extraction, Western Blot, Control, Saline

Journal: The FASEB Journal

Article Title: Ubiquitin‐specific peptidase 7 (USP7) and USP10 mediate deubiquitination of human NHE3 regulating its expression and activity

doi: 10.1096/fj.202001875r

Figure Lengend Snippet: FIGURE 1 USP7 and USP10 are NHE3 binding DUBs. A, The HA-UbVME probe was used to identify DUBs interacting with NHE3. Post-nuclear supernatant (PNS) of Caco-2bbe/NHE3 and SK-CO15 cells were incubated HA-UbVME with or without NEM, which inhibits the covalent linkage between DUBs and HA-UbVME. Immunoblotting using anti-HA antibody showed the presence of NEM-sensitive 110 and 130 kDa bands (arrows) in both cell lines. B, PNS of Caco-2bbe/NHE3 bound to HA-UbVME was immunoblotted with anti-HA, anti-USP10 or anti-USP7 antibody. C, USP7 and USP10 co-immunoprecipitate with NHE3. Caco-2bbe/NHE3 lysate was immunoprecipitated with anti-VSVG antibody P5D4 or IgG as a control, followed by immunoblotting using anti-USP7, anti-USP10, or anti-NHE3 antibody. Lower four panels show immunoblotting of cell lysates. D, USP7, USP10 and NHE3 are present in early endosomes of Caco-2bbe cells. Early endosomal and cytosomal fractions were immunoblotted using antibodies to the proteins indicated. Rab5a and HSP70 were used as a marker for early endosome and cytoplasm, respectively. Representative blots from three or more independent experiments are shown

Article Snippet: The following chemicals were used: HA-tagged ubiquitin-vinyl methyl ester, HA-UbVME (Enzo Life Science, Farmingdale, NY), forskolin (FSK) (Sigma-Aldrich), USP7 inhibitor HBX41108 (Tocris, Minneapolis, MN), USP10 inhibitor spautin-1 (Cayman, Ann Arbor, MI), cycloheximide (Sigma-Aldrich).

Techniques: Binding Assay, Incubation, Western Blot, Immunoprecipitation, Control, Marker

Journal: The FASEB Journal

Article Title: Ubiquitin‐specific peptidase 7 (USP7) and USP10 mediate deubiquitination of human NHE3 regulating its expression and activity

doi: 10.1096/fj.202001875r

Figure Lengend Snippet: FIGURE 2 Knockdown of USP7 or USP10 increases ubiquitination levels of NHE3. A, USP7 and USP10 were knocked down using lentiviral shUSP7 and shUSP10, respectively. Non-targeting scrambled shCon was used as a control. Expression levels of USP7, USP10, and Nedd4-2 were determined. B, Ubiquitination of NHE3 in Caco-2bbe/NHE3 cells was determined. The upper panel shows representative blots of NHE3 ubiquitination and immunoprecipitated NHE3 protein from five independent experiments. Lower, quantitation of Ub levels relative to the amount of immunoprecipitated NHE3 proteins. **, P < .01 compared with shCon. C, Ubiquitination level of endogenous NHE3 in SK-CO15 cells was determined. n = 4. *, P < .05 compared with shCon

Article Snippet: The following chemicals were used: HA-tagged ubiquitin-vinyl methyl ester, HA-UbVME (Enzo Life Science, Farmingdale, NY), forskolin (FSK) (Sigma-Aldrich), USP7 inhibitor HBX41108 (Tocris, Minneapolis, MN), USP10 inhibitor spautin-1 (Cayman, Ann Arbor, MI), cycloheximide (Sigma-Aldrich).

Techniques: Knockdown, Ubiquitin Proteomics, Control, Expressing, Immunoprecipitation, Quantitation Assay

Journal: The FASEB Journal

Article Title: Ubiquitin‐specific peptidase 7 (USP7) and USP10 mediate deubiquitination of human NHE3 regulating its expression and activity

doi: 10.1096/fj.202001875r

Figure Lengend Snippet: FIGURE 3 Knockdown of USP7 or USP10 decreased NHE3 abundance. NHE3 expression in the apical membrane (S-NHE3) of Caco- 2bbe/NHE3 cells (A) or SK-CO15 cells (B) was determined by surface biotinylation as described in Methods and Materials. Total cellular NHE3 (T-NHE3), USP7, and USP10 expression in cell lysate was determined. Representative immunoblots from three independent experiments are shown. Lower charts show the quantitation of S-NHE3 and T-NHE3 relative to β-actin for each cell line. Results are presented as mean ± SEM. n = 3. *, P < .05, **, P < .01 versus shCon. C, NHE3 cellular localization in Caco-2bbe/NHE3 cells was analyzed by confocal microscopy. NHE3 (green) was immunostained using rabbit anti-VSVG antibody. Wheat germ agglutinin (WGA, red) was used as a marker for the apical membrane. Scale bar, 10 µm

Article Snippet: The following chemicals were used: HA-tagged ubiquitin-vinyl methyl ester, HA-UbVME (Enzo Life Science, Farmingdale, NY), forskolin (FSK) (Sigma-Aldrich), USP7 inhibitor HBX41108 (Tocris, Minneapolis, MN), USP10 inhibitor spautin-1 (Cayman, Ann Arbor, MI), cycloheximide (Sigma-Aldrich).

Techniques: Knockdown, Expressing, Membrane, Western Blot, Quantitation Assay, Confocal Microscopy, Marker

Journal: The FASEB Journal

Article Title: Ubiquitin‐specific peptidase 7 (USP7) and USP10 mediate deubiquitination of human NHE3 regulating its expression and activity

doi: 10.1096/fj.202001875r

Figure Lengend Snippet: FIGURE 4 Knockdown of USP7 or USP10 decreased NHE3 abundance. The effect of USP7 (A) or USP10 (B) knockdown on NHE3 activity was determined in Caco-2bbe/NHE3 cells. NHE3 activity was determined as Na+-dependent intracellular pH recovery. Results are presented as mean ± SEM. n ≥ 10. *, P < .05, **, P < .01 compared with shCon

Article Snippet: The following chemicals were used: HA-tagged ubiquitin-vinyl methyl ester, HA-UbVME (Enzo Life Science, Farmingdale, NY), forskolin (FSK) (Sigma-Aldrich), USP7 inhibitor HBX41108 (Tocris, Minneapolis, MN), USP10 inhibitor spautin-1 (Cayman, Ann Arbor, MI), cycloheximide (Sigma-Aldrich).

Techniques: Knockdown, Activity Assay

Journal: The FASEB Journal

Article Title: Ubiquitin‐specific peptidase 7 (USP7) and USP10 mediate deubiquitination of human NHE3 regulating its expression and activity

doi: 10.1096/fj.202001875r

Figure Lengend Snippet: FIGURE 5 USP7 and USP10 have additive effects on NHE3. A, Caco-2bbe/NHE3 cells were treated with the USP7 inhibitor HBX41108 (5 μM), the USP10 inhibitor spautin-1 (10 μM), or DMSO as a control for 24 hours. NHE3 cellular expression was determined. The chart in the bottom shows the quantitation of NHE3 protein relative to β-actin. *, P < .05 versus DMSO. #, P < .05 versus HBX4118, or spautin-1 alone. n = 3. B, The effects of shUSP7 + spautin-1 or shUSP10 + HBX4118 on NHE3 protein expression were determined. *, P < .05, **, P < .01, ***, P < .001 versus DMSO. #, P < .05, ##, P < .01 versus shUSP7, or shUSP10 alone. n = 3

Article Snippet: The following chemicals were used: HA-tagged ubiquitin-vinyl methyl ester, HA-UbVME (Enzo Life Science, Farmingdale, NY), forskolin (FSK) (Sigma-Aldrich), USP7 inhibitor HBX41108 (Tocris, Minneapolis, MN), USP10 inhibitor spautin-1 (Cayman, Ann Arbor, MI), cycloheximide (Sigma-Aldrich).

Techniques: Control, Expressing, Quantitation Assay

Journal: The FASEB Journal

Article Title: Ubiquitin‐specific peptidase 7 (USP7) and USP10 mediate deubiquitination of human NHE3 regulating its expression and activity

doi: 10.1096/fj.202001875r

Figure Lengend Snippet: FIGURE 6 Colocalization of NHE3 with Rab5a and Rab7 is increased by knockdown of USP7 or USP10. A, Expression levels of Rab5a, EE1, and Rab7 were determined in shUSP7 and shUPS10 transduced cells. B, Expression of NHE3 (green) and Rab5a (red) in Caco-2bbe/NHE3 cells was assessed by confocal immunofluorescence microscopy. DAPI was used for nuclear counterstaining (blue). Representative images from three or more independent experiments are shown. Bars, 10 µm. Insets shown magnified view of the boxed regions. Scale bar, 10 μm. C, Graphs represent Pearson’s coefficient of colocalization of NHE3 and Rab5a from more than 22 independent fields of cells. ***, P < .001 versus shCon. D, Western blot shows Rab5a co-immunoprecipitated with NHE3. right, Rab5 expression normalized to β-actin. *, P < .05 versus shCon. E, NHE3 (green) and Rab7 (red) in Caco-2bbe/NHE3 cells were visualized by confocal immunofluorescence microscopy. DAPI (blue), nuclei. Bars, 10 µm. F, Pearson’s coefficient of colocalization of NHE3 and Rab7 from more than 20 independent fields of cells is shown. *, P < .05 versus shCon

Article Snippet: The following chemicals were used: HA-tagged ubiquitin-vinyl methyl ester, HA-UbVME (Enzo Life Science, Farmingdale, NY), forskolin (FSK) (Sigma-Aldrich), USP7 inhibitor HBX41108 (Tocris, Minneapolis, MN), USP10 inhibitor spautin-1 (Cayman, Ann Arbor, MI), cycloheximide (Sigma-Aldrich).

Techniques: Knockdown, Expressing, Immunofluorescence, Microscopy, Western Blot, Immunoprecipitation

Journal: The FASEB Journal

Article Title: Ubiquitin‐specific peptidase 7 (USP7) and USP10 mediate deubiquitination of human NHE3 regulating its expression and activity

doi: 10.1096/fj.202001875r

Figure Lengend Snippet: FIGURE 8 Forskolin (FSK) reduces the interaction of DUBs with NHE3 and potentiates NHE3 ubiquitination. A, NHE3 ubiquitination in response to FSK treatment was determined. The bottom panel shows immunoprecipitated NHE3 proteins. A representative Western immunoblot from four independent experiments is shown. B, Co-immunoprecipitation of USP7 and USP10 with NHE3 was determined in cells treated with FSK. Top two rows show co-immunoprecipitated USP7 and USP10. Third row shows immunoprecipitated NHE3. Lower panels show expression levels of USP7 and USP10 in cells treated with FSK. β-actin was used a loading control. The chart on the left shows the quantitation of co- immunoprecipitated USP7 or USP10 relative to immunoprecipitated NHE3. *, P < .05, **, P < .01 versus 0 minute. n = 3. C, NHE3 ubiquitination was determined in cells treated with FSK. NHE3 was immunoprecipitated from various Caco-2bbe/NHE3 cell lines transiently expressing HA-Ub, and Ub levels were determined using anti-HA antibody. The bottom panel shows immunoprecipitated NHE3 proteins. A representative Western immunoblot from three independent experiments is shown. D, The effect of FSK with or without shUSP10 on NHE3 activity was determined in Caco-2bbe/NHE3 cells. NHE3 activity was determined as Na+-dependent intracellular pH recovery as described in Methods and Materials. Results are presented as mean ± SEM. n ≥ 10. *, P < .05, **, P < .01

Article Snippet: The following chemicals were used: HA-tagged ubiquitin-vinyl methyl ester, HA-UbVME (Enzo Life Science, Farmingdale, NY), forskolin (FSK) (Sigma-Aldrich), USP7 inhibitor HBX41108 (Tocris, Minneapolis, MN), USP10 inhibitor spautin-1 (Cayman, Ann Arbor, MI), cycloheximide (Sigma-Aldrich).

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Expressing, Control, Quantitation Assay, Activity 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 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: 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