nox4  (Proteintech)

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: NOX4 mediates ferroptosis and pain hypersensitivity in SNI mice. (A-B) Western blot analysis of NOX4 expression in the spinal cord following treatment with Bach1-IN-1 or siBach1 (n = 6 per group). (C) Behavioral assessment of PWT and PWL in mice treated with siNOX4 and the ferroptosis inducer RSL3 (n = 9 per group). (D-I) Representative Western blot images and quantification of NOX4, GPX4, SLC7A11, ACSL4, and 4-HNE in the spinal cord of mice receiving the indicated treatments (n = 6 per group). (J-L) Biochemical analysis of Fe 2+ , MDA, and GSH levels in spinal cord (n = 6 per group). (M) TEM analysis of mitochondrial ultrastructure in spinal cord neurons. Scale bar: 500 nm (n = 3 per group). Data analysis used one-way or two-way ANOVA followed by Bonferroni's post-hoc tests. Data are mean ± SEM (∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001 vs. Sham + Vehicle group or Sham + siNC group or Sham + siNC + Vehicle group; # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.0001 vs. SNI + Vehicle group or SNI + siNC group or SNI + siNC + Vehicle group; & P < 0.05, && P < 0.01, &&& P < 0.001, &&&& P < 0.0001 vs. SNI + siNOX4 + 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: Western Blot, Expressing

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: Bach1 transcriptionally activates NOX4 to promote ferroptosis in the spinal cord. (A) Schematic of the potential Bach1-binding site on the NOX4 promoter, predicted using the JASPAR database. (B) Dual-luciferase reporter assay in N2a cells transfected with wild-type (WT) or mutant (MUT) NOX4 promoter constructs and Bach1 overexpression plasmids (∗∗∗∗ P < 0.0001, n = 3 per group). (C) ChIP-qPCR confirming the direct binding of Bach1 to the specific region of the NOX4 promoter (∗∗∗∗ P < 0.0001, n = 3 per group). (D) Representative fluorescence images showing EGFP expression in the spinal dorsal horn, confirming successful AAV transduction. Scale bar: 100 μm. (E) Behavioral assessments showing that AAV-mediated Bach1 overexpression reduced the PWT and PWL, and these effects were reversed by GLX351322 treatment (n = 6 per group). (F-J) Representative Western blots and quantitative analysis of Bach1, NOX4, SLC7A11, and GPX4 protein levels in the spinal cord. AAV-Bach1 increased NOX4 and decreased GPX4 and SLC7A11 expression, which were rescued by GLX351322 (n = 6 per group). (K–N) Biochemical assays showing that AAV-Bach1 elevated the levels of ROS, Fe 2+ , and MDA, and reduced GSH content in spinal cord tissues, and these changes were attenuated by GLX351322 (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 (E-N: ∗∗ 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-Bach1 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: Binding Assay, Luciferase, Reporter Assay, Transfection, Mutagenesis, Construct, Over Expression, ChIP-qPCR, Fluorescence, Expressing, Transduction, Western Blot, 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: 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