gapdh 5174 proteintech usp7  (Proteintech)

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: 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: Subsequently, cells were incubated overnight at 4 °C with USP7 primary antibody (Proteintech, Cat# 66514-1-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: Subsequently, cells were incubated overnight at 4 °C with USP7 primary antibody (Proteintech, Cat# 66514-1-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: Subsequently, cells were incubated overnight at 4 °C with USP7 primary antibody (Proteintech, Cat# 66514-1-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: Subsequently, cells were incubated overnight at 4 °C with USP7 primary antibody (Proteintech, Cat# 66514-1-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