usp7 inhibitor p005091  (MedChemExpress)

Journal: Redox Biology

Article Title: A spinal USP7-Bach1 positive feedback loop drives NOX4-mediated ferroptosis in neuropathic pain

doi: 10.1016/j.redox.2026.104153

Figure Lengend Snippet: Inhibition of USP7 alleviates Bach1 expression, ferroptosis and neuropathic pain. (A) Representative Western blots and quantitative analysis showing increased expression of USP7 in the spinal cord of SNI mice compared with sham controls (∗∗ P < 0.01, ∗∗∗ P < 0.001, n = 6 per group). (B) Double immunofluorescence staining of USP7 (red) with the cellular markers GFAP (astrocytes, green), Iba1 (microglia, green), and NeuN (neurons, green) in the spinal dorsal horn of SNI mice (n = 3 per group). White boxes indicate representative cells shown at higher magnification. Scale bar: 100 μm. (C) Behavioral assessments showing that intraperitoneal administration of the USP7 inhibitor P005091 significantly increased PWT and PWL in SNI mice compared with the SNI + Vehicle group (n = 6 per group). (D-I) Representative Western blots and quantitative analysis showing that P005091 treatment downregulated the expression of USP7, Bach1, and NOX4, and upregulated the expression of GPX4 and SLC7A11 in the spinal cord of SNI mice (n = 6 per group). (J-L) Biochemical assays showing that P005091 treatment reduced the SNI-induced elevations in Fe 2+ and MDA levels, and restored the content of GSH in spinal cord tissues (n = 6 per group). (M) Representative TEM images and quantification of spinal cord mitochondria. Scale bar: 500 nm. Data are presented as mean ± SEM. Significance was determined by one-way or two-way ANOVA followed by Bonferroni's post-hoc tests (∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001 vs. Sham + Vehicle group; # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.0001 vs. SNI + Vehicle group). The value of n represents the number of independent biological samples. All molecular experiments were independently repeated at least three times with consistent results.

Article Snippet: In in vivo experiments, the ferroptosis inhibitor FER-1 (10 mg/kg, i.p., Hy-100579) [ ], the Bach1 inhibitor Bach1-IN-1 (also known as HPPE, 10 mg/kg, i.g., HY-153040 , MCE, China), the NOX4 inhibitor GLX351322 (5 mg/kg, i.p., HY-100111, MCE, China), and the USP7 inhibitor P005091 (15 mg/kg, i.p., HY-15667 , MCE, China) were administered once daily for 5 consecutive days.

Techniques: Inhibition, Expressing, Western Blot, Double Immunofluorescence Staining

Journal: Redox Biology

Article Title: A spinal USP7-Bach1 positive feedback loop drives NOX4-mediated ferroptosis in neuropathic pain

doi: 10.1016/j.redox.2026.104153

Figure Lengend Snippet: USP7 stabilizes Bach1 and is required for its pro-ferroptotic activity in vivo. (A) Representative immunofluorescence images showing co-localization of USP7 (red) and Bach1 (green) in the spinal dorsal horn of mice. Scale bar: 100 μm. (B–C) Exogenous Co-IP analysis in N2a cells co-expressing FLAG-Bach1 and His-USP7 confirmed their specific interaction. (D) Endogenous Co-IP in spinal cord tissue using anti-USP7 antibody demonstrated that Bach1 co-precipitates with USP7 under physiological conditions. (E) Denaturing ubiquitination assay showing that USP7 overexpression markedly reduced HA-Ub-labeled Bach1 in N2a cells following MG132 treatment. (F) Quantification of CHX chase assay results demonstrates that USP7 overexpression extends the half-life of Bach1 from 3.43 h to 10.68 h, as shown by the representative immunoblot of Bach1 protein levels at 0, 2, 4, and 6 h (∗∗∗∗ P < 0.0001, n = 3 per group). (G) Representative EGFP fluorescence images confirming successful viral transduction in spinal dorsal horn. Scale bar: 100 μm. (H) Behavioral assessments of PWT and PWL (n = 9 per group). siUSP7 alleviated SNI-induced pain hypersensitivity, whereas Bach1 overexpression reversed this protective effect. (I-Q) Western blot analysis and quantification of USP7, Bach1, NOX4, GPX4, SLC7A11, ACSL4, and 4-HNE expression in spinal cord from indicated groups (n = 6 per group). (R–U) Biochemical measurements of Fe 2+ , MDA, GSH, and ATP levels in spinal cord tissues (n = 6 per group). (V) Representative TEM images and quantification showing mitochondrial morphology in spinal neurons (n = 3 per group). Scale bar: 500 nm. Data are presented as mean ± SEM. Comparisons between two independent groups were performed using unpaired two-tailed Student's t-tests. All multi-group comparisons were conducted using one-way or two-way ANOVA followed by Bonferroni's post-hoc tests (H–V: ∗ P < 0.05, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001 vs. Sham + AAV-Vector + siNC group; # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.0001 vs. SNI + AAV-Vector + siNC group; & P < 0.05, && P < 0.01, &&& P < 0.001, &&&& P < 0.0001 vs. SNI + AAV-Vector + siUSP7 group). The value of n represents the number of independent biological samples. All molecular and cellular experiments were independently repeated at least three times with consistent results.

Article Snippet: In in vivo experiments, the ferroptosis inhibitor FER-1 (10 mg/kg, i.p., Hy-100579) [ ], the Bach1 inhibitor Bach1-IN-1 (also known as HPPE, 10 mg/kg, i.g., HY-153040 , MCE, China), the NOX4 inhibitor GLX351322 (5 mg/kg, i.p., HY-100111, MCE, China), and the USP7 inhibitor P005091 (15 mg/kg, i.p., HY-15667 , MCE, China) were administered once daily for 5 consecutive days.

Techniques: Activity Assay, In Vivo, Immunofluorescence, Co-Immunoprecipitation Assay, Expressing, Ubiquitin Proteomics, Over Expression, Labeling, Western Blot, Fluorescence, Transduction, Two Tailed Test, Plasmid Preparation

Journal: Redox Biology

Article Title: A spinal USP7-Bach1 positive feedback loop drives NOX4-mediated ferroptosis in neuropathic pain

doi: 10.1016/j.redox.2026.104153

Figure Lengend Snippet: USP7 overexpression induces Bach1-dependent ferroptosis and pain hypersensitivity in naïve mice. (A) Representative fluorescence images showing EGFP expression in the spinal dorsal horn, confirming successful AAV-USP7 transduction. Scale bar: 100 μm. (B) Behavioral assessments showing that AAV-USP7 significantly decreased the PWT and PWL in naïve mice compared with the AAV-Vector control group (n = 6 per group). (C) RT-qPCR analysis of Bach1 mRNA levels in spinal tissues following USP7 overexpression (n = 6 per group). (D-F) Representative Western blots and quantitative analysis of spinal cord tissues showing increased protein levels of USP7 and Bach1 following AAV-USP7 injection (n = 6 per group). (G-H) Behavioral tests demonstrating that Bach1-IN-1 treatment reversed the reductions in PWT and PWL induced by AAV-USP7 overexpression (n = 6 per group). (I-L) Representative Western blots and quantitative analysis showing that AAV-USP7 increased NOX4 expression and decreased the levels of SLC7A11 and GPX4, which were restored by Bach1-IN-1 treatment (n = 6 per group). (M − O) Biochemical assays quantifying ferroptosis-related indicators in spinal cord tissues. AAV-USP7 elevated Fe 2+ and MDA levels and reduced GSH content, and these changes were attenuated by Bach1-IN-1 (n = 6 per group). Data are presented as mean ± SEM. Comparisons between two independent groups were performed using unpaired two-tailed Student's t-tests. All multi-group comparisons were conducted using one-way or two-way ANOVA followed by Bonferroni's post-hoc tests (∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001 vs. Naïve + AAV-Vector group; # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.0001 vs. Naïve + AAV-USP7 group). The value of n represents the number of independent biological samples. All molecular experiments were independently repeated at least three times with consistent results.

Article Snippet: In in vivo experiments, the ferroptosis inhibitor FER-1 (10 mg/kg, i.p., Hy-100579) [ ], the Bach1 inhibitor Bach1-IN-1 (also known as HPPE, 10 mg/kg, i.g., HY-153040 , MCE, China), the NOX4 inhibitor GLX351322 (5 mg/kg, i.p., HY-100111, MCE, China), and the USP7 inhibitor P005091 (15 mg/kg, i.p., HY-15667 , MCE, China) were administered once daily for 5 consecutive days.

Techniques: Over Expression, Fluorescence, Expressing, Transduction, Plasmid Preparation, Control, Quantitative RT-PCR, Western Blot, Injection, Two Tailed Test

Journal: Redox Biology

Article Title: A spinal USP7-Bach1 positive feedback loop drives NOX4-mediated ferroptosis in neuropathic pain

doi: 10.1016/j.redox.2026.104153

Figure Lengend Snippet: USP7 promotes ferroptosis through Bach1 and forms a positive feedback loop with Bach1. (A-F) Representative Western blots and quantitative analysis of USP7, Bach1, NOX4, SLC7A11, and GPX4 protein levels in N2a cells following USP7 overexpression and/or Bach1 inhibition (n = 3 per group). (G) Flow cytometric analysis using the fluorescent probe BODIPY™ 581/591 C11 to detect lipid ROS. The ratio of oxidized (FITC) to total fluorescence was increased in OE-USP7 cells and reduced by co-treatment with Bach1-IN-1 (n = 3 per group). (H-J) Biochemical quantification of ferroptosis-related parameters in cell lysates. OE-USP7 elevated intracellular Fe 2+ and MDA levels and decreased GSH content; these changes were attenuated by Bach1-IN-1 (n = 3 per group). (K) Cell Counting Kit-8 (CCK-8) assay showing that Bach1-IN-1 restored the viability of OE-USP7 cells (n = 3 per group). (L) RT-qPCR analysis showing that USP7 mRNA levels were upregulated by Bach1 overexpression (OE-Bach1) and downregulated by Bach1 knockdown (si-Bach1) (n = 3 per group). (M − O) Representative Western blots and quantification of Bach1 and USP7 protein levels in N2a cells transfected with OE-Bach1 or si-Bach1, confirming the regulatory effect of Bach1 on USP7 expression (n = 3 per group). (P) JASPAR analysis of the USP7 promoter identified two putative Bach1-binding sites (MAREs) located at −1756 bp (Site 1) and −1159 bp (Site 2). (Q) Motif alignment revealed that Site 1 closely matches the consensus Bach1-binding sequence. (R) ChIP-qPCR analysis using primers flanking Site 1 (−1756 bp), confirming enrichment of Bach1 at this specific promoter locus in N2a cells (n = 3 per group). (S) Schematic of wild-type (WT) and mutant (MUT) USP7 promoter constructs used in luciferase reporter assays. (T) Luciferase assays show that Bach1 overexpression increases activity of the wild-type USP7 promoter, while mutation of the binding sites eliminates this effect (n = 3 per group). Data are presented as mean ± SEM. Comparisons between two independent groups were performed using unpaired two-tailed Student's t-tests. All multi-group comparisons were conducted using one-way or two-way ANOVA followed by Bonferroni's post-hoc tests (B–K: ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗∗ P < 0.0001 vs. Ctrl group; # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.0001 vs. OE-USP7 group; L-T:∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001). The value of n represents the number of independent biological samples. All molecular and cellular experiments were independently repeated at least three times with consistent results.

Article Snippet: In in vivo experiments, the ferroptosis inhibitor FER-1 (10 mg/kg, i.p., Hy-100579) [ ], the Bach1 inhibitor Bach1-IN-1 (also known as HPPE, 10 mg/kg, i.g., HY-153040 , MCE, China), the NOX4 inhibitor GLX351322 (5 mg/kg, i.p., HY-100111, MCE, China), and the USP7 inhibitor P005091 (15 mg/kg, i.p., HY-15667 , MCE, China) were administered once daily for 5 consecutive days.

Techniques: Western Blot, Over Expression, Inhibition, Fluorescence, Cell Counting, CCK-8 Assay, Quantitative RT-PCR, Knockdown, Transfection, Expressing, Binding Assay, Sequencing, ChIP-qPCR, Mutagenesis, Construct, Luciferase, Activity Assay, Two Tailed Test

Journal: Journal of translational medicine

Article Title: Ubiquitin-specific protease 7 maintains c-Myc stability to support pancreatic cancer glycolysis and tumor growth.

doi: 10.1186/s12967-024-05962-6

Figure Lengend Snippet: Fig. 3 Hypoxia and extracellular matrix stiffness induces the expression of USP7 in PDAC cells. A PDAC cells (AsPC-1, CFPAC-1 and PANC1) were cultured under normoxic (5% O2) and hypoxic (1% O2) conditions for 24 h. Then, HIF1α transcriptional activity in PDAC cells was measured (n = 3). B The mRNA level of USP7 in groups as indicated in (A) was determined by real-time qPCR analysis (n = 3). C PDAC cells (AsPC-1, CFPAC-1 and PANC1) were cultured under hypoxic (1% O2) conditions for 24 h, with or without treatment of an HIF1α inhibitor LW6. Then, HIF1α transcriptional activity in PDAC cells was measured (n = 3). D The mRNA level of USP7 in groups as indicated in (B) was determined by real-time qPCR analysis (n = 3). E USP7 mRNA levels in PDAC samples from TCGA database were compared with the HIF1α signature using correlation analysis (n = 178). F CFPAC-1 and PANC1 seeded on soft (0.5 kPa) and stiff matrices (12 kPa) 6-well plates and cultured under normoxic (5% O2) or hypoxic (1% O2) conditions for 24 h. The mRNA level of USP7 was determined by real-time qPCR analysis (n = 3) Values were compared by the Student’s t test (A–D), Spearman’s rank correlation methods (E), and one-way ANOVA multiple comparisons with Tukey’s method among groups (F). Experiments were independently repeated three times (A–D, F) with similar results. *P < 0.05 and **P < 0.01; # indicates comparison with Stiff + 1% O2 group, #P < 0.05 and ##P < 0.01

Article Snippet: USP7 inhibitor P5091 (Cat. HY-15667), HIF1α inhibitor HY-13,671 (Cat. HY-13671), and MG132 (Cat. HY-13259) were obtained from MedChemExpress.

Techniques: Expressing, Cell Culture, Activity Assay, Comparison

Journal: Journal of translational medicine

Article Title: Ubiquitin-specific protease 7 maintains c-Myc stability to support pancreatic cancer glycolysis and tumor growth.

doi: 10.1186/s12967-024-05962-6

Figure Lengend Snippet: Fig. 6 Targeting USP7 in a transgenic mouse model of PDAC inhibits tumor progression and glycolytic metabolism.A Treatment schedule of P5091 in the KPC mice (n = 5 per group). B Histological analysis of pancreas lesions upon P5091 treatment in KPC mice. PanINs: pancreatic intraepithelial neoplasia. C IHC analysis of c-Myc, GLUT1, HK2, and LDHA in KPC pancreas lesions upon P5091 treatment. Scale bar: 50 μm. D Quantification data of c-Myc staining in C (n = 5 per group). E Quantification data of GLUT1, HK2, and LDHA staining in C (n = 5 per group) Values were compared by Fisher’s exact test (B) and the Student’s t test (C, E). Animal experiment in this figure was not repeated. *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: USP7 inhibitor P5091 (Cat. HY-15667), HIF1α inhibitor HY-13,671 (Cat. HY-13671), and MG132 (Cat. HY-13259) were obtained from MedChemExpress.

Techniques: Transgenic Assay, Staining

Journal: Journal of translational medicine

Article Title: Ubiquitin-specific protease 7 maintains c-Myc stability to support pancreatic cancer glycolysis and tumor growth.

doi: 10.1186/s12967-024-05962-6

Figure Lengend Snippet: Fig. 7 USP7 expression correlates c-Myc and glycolytic proteins in clinical samples.A Analysis of the correlation between USP7, c-Myc, and glycolytic proteins (GLUT1, HK2, and LDHA) in a PDAC tissue microarray. P values were compared by the Spearman’s rank correlation methods. B The schematic diagram illustrates that the stiffness of the extracellular matrix and a hypoxic microenvironment trigger the expression of USP7. USP7, in turn, boosts the stability of c-Myc by facilitating its ubiquitination. Consequently, c-Myc promotes the transcription and regulation of glycolytic genes, leading to elevated glycolysis levels and fostering tumor growth

Article Snippet: USP7 inhibitor P5091 (Cat. HY-15667), HIF1α inhibitor HY-13,671 (Cat. HY-13671), and MG132 (Cat. HY-13259) were obtained from MedChemExpress.

Techniques: Expressing, Microarray, Ubiquitin Proteomics