rabbit anti usp7 antibody  (Proteintech)

Journal: Cellular & molecular biology letters

Article Title: Role of USP7 in the regulation of tolerogenic dendritic cell function in type 1 diabetes.

doi: 10.1186/s11658-025-00727-5

Figure Lengend Snippet: Fig. 1 Tolerogenic DCs of transgenic NOD.Stat5b-CA mice express higher USP7 than immunogenic DCs of NOD mice. A Representative FACS plots showing gating strategy used for USP7 expression analysis in purified splenic CD11c + DCs from the spleen. B Counter plots showing CD11c+USP7+ cell frequencies in purified splenic DCs of NOD and transgenic NOD.Stat5b-CA mice. C Representative bar graph showing USP7 frequency. D Absolute numbers of USP7 expressing DCs. E Histogram showing USP7 expression level in purified splenic CD11c+ DCs. F Mean fluorescence intensity (MFI) of USP7 expression and (G) Western blot analysis of USP7 expression and β-actin used as a loading control. Data are shown as the mean ± SEM of three independent experiments. The two-tailed unpaired Student’s t-test was used. *P < 0.05; **P < 0.01.****P < 0.0001

Article Snippet: Membranes were then blocked with 5% dry milk and incubated overnight at 4 °C with an anti-USP7 primary antibody (cat# 4833, Cell Signaling Technology), followed by an appropriate secondary antibody (cat# 7074S, Cell Signaling Technology). β-actin (cat# 8457, Cell Signaling Technology) was used as a loading control.

Techniques: Transgenic Assay, Expressing, Purification, Fluorescence, Western Blot, Control, Two Tailed Test

Journal: Cellular & molecular biology letters

Article Title: Role of USP7 in the regulation of tolerogenic dendritic cell function in type 1 diabetes.

doi: 10.1186/s11658-025-00727-5

Figure Lengend Snippet: Fig. 2 Pharmacological inhibition of USP7 increases DC mature phenotype and facilitates their proinflammatory cytokines production. Purified splenic DCs (1 × 105 cells/well) from NOD and NOD. Stat5b-CA mice were cultured in the presence (+ P5091) or absence (− P5091) of USP7 inhibitor (5 µM/mL) for 24 h. DCs were washed, labeled with anti-CD11c mAbs in combination with anti-MHC class II, anti-CD40, anti-CD80, or anti-CD86 mAbs, and analyzed by FACS. A Flow cytometry plots depicted cell surface expression of MHC class II, CD40, CD80, and CD86. B Representative bar graphs showing the percentages of positive cells with respect to the total population of purified splenic DCs. C Mean fluorescence intensities (MFI) values of FACS profiles. For cytokines quantification, purified splenic DCs (5 × 105 cells/well) from NOD and NOD.Stat5b-CA mice were cultured for 24 h in the presence (+ P5091) or absence (− P5091) of USP7 inhibitor (5 µM/mL) and then stimulated with LPS (1 µg/mL) for an additional 24 h. Quantification of TNF-α, IL-1β, IL-6, IL-10, and TGF-β released in the supernatants of splenic DCs before (D–H) and after LPS stimulation (I–M). Data are shown as the mean ± SEM of at least three independent experiments. One-way ANOVA followed by Tukey’s post hoc test was used. n.s, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

Article Snippet: Membranes were then blocked with 5% dry milk and incubated overnight at 4 °C with an anti-USP7 primary antibody (cat# 4833, Cell Signaling Technology), followed by an appropriate secondary antibody (cat# 7074S, Cell Signaling Technology). β-actin (cat# 8457, Cell Signaling Technology) was used as a loading control.

Techniques: Inhibition, Purification, Cell Culture, Labeling, Flow Cytometry, Expressing, Fluorescence

Journal: Cellular & molecular biology letters

Article Title: Role of USP7 in the regulation of tolerogenic dendritic cell function in type 1 diabetes.

doi: 10.1186/s11658-025-00727-5

Figure Lengend Snippet: Fig. 3 USP7 blockade decreases PD-L1 and PD-L2 expression in tolerogenic Stat5b-CA.DCs. Purified splenic DCs (1 × 105 cells/well) from NOD and NOD.Stat5b-CA mice were cultured for 24 h in the presence (+ P5091) or absence (− P5091) of USP7 inhibitor (5 µM/mL). DCs were washed, labeled with anti-CD11c mAbs in combination with anti-PD-L1 and anti-PD-L2 mAbs. A–D Flow cytometry plots depict PD-L1 and PD-L2 expression (A, C) and cell frequencies (B, D) in CD11c+ DCs. E Flow cytometry histogram depicts levels of PD-L1 and PD-L2 expressions and (F–G) MFI in CD11c+ DCs treated with or without USP7 inhibitor (5 µM/mL). Data are shown as the mean ± SEM of five independent experiments. One-way ANOVA followed by Tukey’s post hoc test was used. n.s, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

Article Snippet: Membranes were then blocked with 5% dry milk and incubated overnight at 4 °C with an anti-USP7 primary antibody (cat# 4833, Cell Signaling Technology), followed by an appropriate secondary antibody (cat# 7074S, Cell Signaling Technology). β-actin (cat# 8457, Cell Signaling Technology) was used as a loading control.

Techniques: Expressing, Purification, Cell Culture, Labeling, Flow Cytometry

Journal: Cellular & molecular biology letters

Article Title: Role of USP7 in the regulation of tolerogenic dendritic cell function in type 1 diabetes.

doi: 10.1186/s11658-025-00727-5

Figure Lengend Snippet: Fig. 4 Inhibition of USP7 promotes cDC1 over cDC2 in tolerogenic Stat5b-CA.DCs. Purified splenic DCs (1 × 105 cells/well) from NOD and NOD.Stat5b-CA mice were cultured in the presence (+ P5091) or absence (− P5091) of USP7 inhibitor (5 µM/mL) for 24 h. DCs were washed, labeled with anti-CD11c mAbs in combination with anti-CD11b, anti-Sirpα, and anti-XCR1 mAbs. A, C, E FACS profiles showing CD11b, XCR1, and Sirpa expression in splenic CD11c+ DCs treated with or without USP7 inhibitor. B, D, F Representative bar graphs of CD11c+CD11b+, CD11b+Sirpa+, CD11b−XCR1+ cDC frequencies. (G, I, and K) Representative histograms of CD11b, Sirpα, and XCR1 gated on CD11c+ as assessed by flow cytometry. H, J, L Mean fluorescence intensity (MFI) values of CD11b, Sirpα (gated on CD11c+CD11b+ DCs), and XCR1 (gated on CD11c+CD11b− DCs) expression. Data are shown as the mean ± SEM of at least four independent experiments. One-way ANOVA followed by Tukey’s post hoc test was used. n.s, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

Article Snippet: Membranes were then blocked with 5% dry milk and incubated overnight at 4 °C with an anti-USP7 primary antibody (cat# 4833, Cell Signaling Technology), followed by an appropriate secondary antibody (cat# 7074S, Cell Signaling Technology). β-actin (cat# 8457, Cell Signaling Technology) was used as a loading control.

Techniques: Inhibition, Purification, Cell Culture, Labeling, Expressing, Flow Cytometry, Fluorescence

Journal: Cellular & molecular biology letters

Article Title: Role of USP7 in the regulation of tolerogenic dendritic cell function in type 1 diabetes.

doi: 10.1186/s11658-025-00727-5

Figure Lengend Snippet: Fig. 5 Blockade of USP7 downregulates IRF4 and upregulates IRF8 expression. Purified splenic cDCs (1 × 105 cells/well) from NOD and NOD.Stat5b-CA mice were cultured for 24 h in the presence (+ P5091) or absence (− P5091) of USP7 inhibitor (5 µM/mL). cDCs were washed, labeled with anti-CD11c mAbs in combination with Abs anti-IRF-4 or anti-IRF-8, and analyzed by FACS. A, C Counter plots (left) and representative bar graphs (right) showing CD11c+IRF4+ (A) and CD11c+IRF8+ (C) cell frequencies in purified splenic cDCs. B, D Flow cytometry histogram showing IRF4 (B) and IRF8 expressions (D) in splenic CD11c+ DCs. E, G Flow cytometry histograms showing IRF4 (E) and IRF8 (G) expression in purified splenic CD11c+ DCs treated with or without USP7 inhibitor (5 µM/mL) for 24 h. F, H Mean fluorescence intensity (MFI) values of IRF4 (F) and IRF8 (H) expression. Data are shown as the mean ± SEM of five independent experiments. The significance was calculated using One-way ANOVA with Tukey’s post-test. n.s, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

Article Snippet: Membranes were then blocked with 5% dry milk and incubated overnight at 4 °C with an anti-USP7 primary antibody (cat# 4833, Cell Signaling Technology), followed by an appropriate secondary antibody (cat# 7074S, Cell Signaling Technology). β-actin (cat# 8457, Cell Signaling Technology) was used as a loading control.

Techniques: Expressing, Purification, Cell Culture, Labeling, Flow Cytometry, Fluorescence

Journal: Cellular & molecular biology letters

Article Title: Role of USP7 in the regulation of tolerogenic dendritic cell function in type 1 diabetes.

doi: 10.1186/s11658-025-00727-5

Figure Lengend Snippet: Fig. 6 USP7 controls IRF4 and IRF8 expression by regulating Ezh2. A Crystal structure of overall protein– protein binding between USP7 and Ezh2. B Ribbon representation of the crystal structure of USP7:Ezh2 peptide complex with the USP7 peptide represented by blue color and Ezh2 peptide represented by red color (left). The interactions formed between USP7 and the Ezh2 peptide are shown as yellow dashed lines. The residues involved in the interactions are labeled (right) and presented in table form. C–G Splenic DCs purified from NOD and NOD.Stat5b-CA mice were incubated with either vehicle (0.1% DMSO) or with the Ezh2 inhibitor GSK343 (3 μM/mL) alone or together with the USP7 inhibitor P5091 (5 μM/mL) for 24 h. Thereafter, cells were washed and then stained with anti-CD11c, anti-Ezh2, anti-IRF4, and anti-IRF8 antibodies and analyzed by flow cytometry. C, D Histogram profile (left panel) and Mean fluorescence intensity (MFI) quantification (right panel) of the level of Ezh2 expression in CD11c+ DCs. Data are shown as the mean ± SEM of three independent experiments. The two-tailed unpaired Student’s t-test was used. **P < 0.01. E MFI values of Ezh2 in DCs treated with either vehicle or P5091 inhibitor. F, G MFI values of IRF4 and IRF8 in vehicle-treated DCs or DCs treated with Ezh2 inhibitor alone or together with P5091 inhibitor. Data are shown as the mean ± SEM of three independent experiments. One-way ANOVA followed by Tukey’s post hoc test was used. n.s, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

Article Snippet: Membranes were then blocked with 5% dry milk and incubated overnight at 4 °C with an anti-USP7 primary antibody (cat# 4833, Cell Signaling Technology), followed by an appropriate secondary antibody (cat# 7074S, Cell Signaling Technology). β-actin (cat# 8457, Cell Signaling Technology) was used as a loading control.

Techniques: Expressing, Protein Binding, Labeling, Purification, Incubation, Staining, Flow Cytometry, Fluorescence, Two Tailed Test

Journal: Cellular & molecular biology letters

Article Title: Role of USP7 in the regulation of tolerogenic dendritic cell function in type 1 diabetes.

doi: 10.1186/s11658-025-00727-5

Figure Lengend Snippet: Fig. 7 Inhibition of USP7 in tolerogenic Stat5b-CA.DCs reduce Tregs and Th2 responses and promote IL-17-producing Th17 cell subset. Purified splenic DCs from NOD and NOD.Stat5b-CA mice were pre-incubated with (+ PD5091) or without (− PD509) USP7 inhibitor (5 µM/mL) for 24 h and then stimulated with LPS (1 µg/mL) for additional 24 h. Cells were then washed, and 6 × 106 cells were i.v injected into 8–10 weeks old NOD mice. After 7 days, spleen cells were harvested and analyzed for Tregs and Th1/Th2/Th17 cell subsets and their cytokine profiles by FACS. A–H Representative FACS profile and percentage of CD4+Foxp3+ Tregs (A, B), CD4+Gata-3+ Th2 (C-D), CD4+RORγt+ Th17 (E, F) and CD4+T-bet+ Th1 (G, H) cells. I–L Splenic cells of recipient NOD mice were stained with different T cell subsets and cytokine antibodies and analyzed by FACS. Percentages of Foxp3+IL-10+ (I), CD4+IL-4+ (J), CD4+IL-17+ (K), and CD4+IFN-γ+ (L) T cell subsets. In all experiments, 4 mice per group were used, and the results are expressed as mean ± SEM. The significance was calculated using One-way ANOVA with Tukey’s post-test. n.s., not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

Article Snippet: Membranes were then blocked with 5% dry milk and incubated overnight at 4 °C with an anti-USP7 primary antibody (cat# 4833, Cell Signaling Technology), followed by an appropriate secondary antibody (cat# 7074S, Cell Signaling Technology). β-actin (cat# 8457, Cell Signaling Technology) was used as a loading control.

Techniques: Inhibition, Purification, Incubation, Injection, Staining

Journal: Cellular & molecular biology letters

Article Title: Role of USP7 in the regulation of tolerogenic dendritic cell function in type 1 diabetes.

doi: 10.1186/s11658-025-00727-5

Figure Lengend Snippet: Fig. 8 USP7 blockade halts the capacity of tolerogenic Stat5b-CA.DCs to protect NOD mice from diabetes. Purified splenic DC of NOD and NOD.Stat5b-CA mice were treated or not with USP7 inhibitor P5091 (5 µM/mL, for 24 h), stimulated with LPS for another 24 h, and intravenously injected into 3–4 weeks old female NOD mice (7 mice per group, 6 × 106 cells/mouse). Recipient NOD mice were followed for diabetes development until 32 weeks of age. For comparison of diabetes incidence between different groups, the Gehan–Breslow–Wilcoxon test was performed to calculate the significant difference. n.s., not significant; ****P < 0.0001

Article Snippet: Membranes were then blocked with 5% dry milk and incubated overnight at 4 °C with an anti-USP7 primary antibody (cat# 4833, Cell Signaling Technology), followed by an appropriate secondary antibody (cat# 7074S, Cell Signaling Technology). β-actin (cat# 8457, Cell Signaling Technology) was used as a loading control.

Techniques: Purification, Injection, Comparison

Journal: Cellular & molecular biology letters

Article Title: Role of USP7 in the regulation of tolerogenic dendritic cell function in type 1 diabetes.

doi: 10.1186/s11658-025-00727-5

Figure Lengend Snippet: Fig. 1 Tolerogenic DCs of transgenic NOD.Stat5b-CA mice express higher USP7 than immunogenic DCs of NOD mice. A Representative FACS plots showing gating strategy used for USP7 expression analysis in purified splenic CD11c + DCs from the spleen. B Counter plots showing CD11c+USP7+ cell frequencies in purified splenic DCs of NOD and transgenic NOD.Stat5b-CA mice. C Representative bar graph showing USP7 frequency. D Absolute numbers of USP7 expressing DCs. E Histogram showing USP7 expression level in purified splenic CD11c+ DCs. F Mean fluorescence intensity (MFI) of USP7 expression and (G) Western blot analysis of USP7 expression and β-actin used as a loading control. Data are shown as the mean ± SEM of three independent experiments. The two-tailed unpaired Student’s t-test was used. *P < 0.05; **P < 0.01.****P < 0.0001

Article Snippet: Thereafter, cells were collected, washed with PBS, fixed with 4% paraformaldehyde (PFA) for 40 min, and then permeabilized using the Foxp3 Staining Kit (eBioscience, San Diego, California, USA) for 35 min. After permeabilization, cells were incubated with anti-IRF4-PE (clone 3E4; cat# 12–9858-82), anti-IRF8APC (clone V3GYWCH; cat# 2093671), or with anti-Ezh2 (cat# 4905S, Cell Signaling Technology), and anti-USP7 primary Ab (cat# 4833, Cell Signaling Technology, Whitby, Ontario, Canada).

Techniques: Transgenic Assay, Expressing, Purification, Fluorescence, Western Blot, Control, Two Tailed Test

Journal: Cellular & molecular biology letters

Article Title: Role of USP7 in the regulation of tolerogenic dendritic cell function in type 1 diabetes.

doi: 10.1186/s11658-025-00727-5

Figure Lengend Snippet: Fig. 2 Pharmacological inhibition of USP7 increases DC mature phenotype and facilitates their proinflammatory cytokines production. Purified splenic DCs (1 × 105 cells/well) from NOD and NOD. Stat5b-CA mice were cultured in the presence (+ P5091) or absence (− P5091) of USP7 inhibitor (5 µM/mL) for 24 h. DCs were washed, labeled with anti-CD11c mAbs in combination with anti-MHC class II, anti-CD40, anti-CD80, or anti-CD86 mAbs, and analyzed by FACS. A Flow cytometry plots depicted cell surface expression of MHC class II, CD40, CD80, and CD86. B Representative bar graphs showing the percentages of positive cells with respect to the total population of purified splenic DCs. C Mean fluorescence intensities (MFI) values of FACS profiles. For cytokines quantification, purified splenic DCs (5 × 105 cells/well) from NOD and NOD.Stat5b-CA mice were cultured for 24 h in the presence (+ P5091) or absence (− P5091) of USP7 inhibitor (5 µM/mL) and then stimulated with LPS (1 µg/mL) for an additional 24 h. Quantification of TNF-α, IL-1β, IL-6, IL-10, and TGF-β released in the supernatants of splenic DCs before (D–H) and after LPS stimulation (I–M). Data are shown as the mean ± SEM of at least three independent experiments. One-way ANOVA followed by Tukey’s post hoc test was used. n.s, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

Article Snippet: Thereafter, cells were collected, washed with PBS, fixed with 4% paraformaldehyde (PFA) for 40 min, and then permeabilized using the Foxp3 Staining Kit (eBioscience, San Diego, California, USA) for 35 min. After permeabilization, cells were incubated with anti-IRF4-PE (clone 3E4; cat# 12–9858-82), anti-IRF8APC (clone V3GYWCH; cat# 2093671), or with anti-Ezh2 (cat# 4905S, Cell Signaling Technology), and anti-USP7 primary Ab (cat# 4833, Cell Signaling Technology, Whitby, Ontario, Canada).

Techniques: Inhibition, Purification, Cell Culture, Labeling, Flow Cytometry, Expressing, Fluorescence

Journal: Cellular & molecular biology letters

Article Title: Role of USP7 in the regulation of tolerogenic dendritic cell function in type 1 diabetes.

doi: 10.1186/s11658-025-00727-5

Figure Lengend Snippet: Fig. 3 USP7 blockade decreases PD-L1 and PD-L2 expression in tolerogenic Stat5b-CA.DCs. Purified splenic DCs (1 × 105 cells/well) from NOD and NOD.Stat5b-CA mice were cultured for 24 h in the presence (+ P5091) or absence (− P5091) of USP7 inhibitor (5 µM/mL). DCs were washed, labeled with anti-CD11c mAbs in combination with anti-PD-L1 and anti-PD-L2 mAbs. A–D Flow cytometry plots depict PD-L1 and PD-L2 expression (A, C) and cell frequencies (B, D) in CD11c+ DCs. E Flow cytometry histogram depicts levels of PD-L1 and PD-L2 expressions and (F–G) MFI in CD11c+ DCs treated with or without USP7 inhibitor (5 µM/mL). Data are shown as the mean ± SEM of five independent experiments. One-way ANOVA followed by Tukey’s post hoc test was used. n.s, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

Article Snippet: Thereafter, cells were collected, washed with PBS, fixed with 4% paraformaldehyde (PFA) for 40 min, and then permeabilized using the Foxp3 Staining Kit (eBioscience, San Diego, California, USA) for 35 min. After permeabilization, cells were incubated with anti-IRF4-PE (clone 3E4; cat# 12–9858-82), anti-IRF8APC (clone V3GYWCH; cat# 2093671), or with anti-Ezh2 (cat# 4905S, Cell Signaling Technology), and anti-USP7 primary Ab (cat# 4833, Cell Signaling Technology, Whitby, Ontario, Canada).

Techniques: Expressing, Purification, Cell Culture, Labeling, Flow Cytometry

Journal: Cellular & molecular biology letters

Article Title: Role of USP7 in the regulation of tolerogenic dendritic cell function in type 1 diabetes.

doi: 10.1186/s11658-025-00727-5

Figure Lengend Snippet: Fig. 4 Inhibition of USP7 promotes cDC1 over cDC2 in tolerogenic Stat5b-CA.DCs. Purified splenic DCs (1 × 105 cells/well) from NOD and NOD.Stat5b-CA mice were cultured in the presence (+ P5091) or absence (− P5091) of USP7 inhibitor (5 µM/mL) for 24 h. DCs were washed, labeled with anti-CD11c mAbs in combination with anti-CD11b, anti-Sirpα, and anti-XCR1 mAbs. A, C, E FACS profiles showing CD11b, XCR1, and Sirpa expression in splenic CD11c+ DCs treated with or without USP7 inhibitor. B, D, F Representative bar graphs of CD11c+CD11b+, CD11b+Sirpa+, CD11b−XCR1+ cDC frequencies. (G, I, and K) Representative histograms of CD11b, Sirpα, and XCR1 gated on CD11c+ as assessed by flow cytometry. H, J, L Mean fluorescence intensity (MFI) values of CD11b, Sirpα (gated on CD11c+CD11b+ DCs), and XCR1 (gated on CD11c+CD11b− DCs) expression. Data are shown as the mean ± SEM of at least four independent experiments. One-way ANOVA followed by Tukey’s post hoc test was used. n.s, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

Article Snippet: Thereafter, cells were collected, washed with PBS, fixed with 4% paraformaldehyde (PFA) for 40 min, and then permeabilized using the Foxp3 Staining Kit (eBioscience, San Diego, California, USA) for 35 min. After permeabilization, cells were incubated with anti-IRF4-PE (clone 3E4; cat# 12–9858-82), anti-IRF8APC (clone V3GYWCH; cat# 2093671), or with anti-Ezh2 (cat# 4905S, Cell Signaling Technology), and anti-USP7 primary Ab (cat# 4833, Cell Signaling Technology, Whitby, Ontario, Canada).

Techniques: Inhibition, Purification, Cell Culture, Labeling, Expressing, Flow Cytometry, Fluorescence

Journal: Cellular & molecular biology letters

Article Title: Role of USP7 in the regulation of tolerogenic dendritic cell function in type 1 diabetes.

doi: 10.1186/s11658-025-00727-5

Figure Lengend Snippet: Fig. 5 Blockade of USP7 downregulates IRF4 and upregulates IRF8 expression. Purified splenic cDCs (1 × 105 cells/well) from NOD and NOD.Stat5b-CA mice were cultured for 24 h in the presence (+ P5091) or absence (− P5091) of USP7 inhibitor (5 µM/mL). cDCs were washed, labeled with anti-CD11c mAbs in combination with Abs anti-IRF-4 or anti-IRF-8, and analyzed by FACS. A, C Counter plots (left) and representative bar graphs (right) showing CD11c+IRF4+ (A) and CD11c+IRF8+ (C) cell frequencies in purified splenic cDCs. B, D Flow cytometry histogram showing IRF4 (B) and IRF8 expressions (D) in splenic CD11c+ DCs. E, G Flow cytometry histograms showing IRF4 (E) and IRF8 (G) expression in purified splenic CD11c+ DCs treated with or without USP7 inhibitor (5 µM/mL) for 24 h. F, H Mean fluorescence intensity (MFI) values of IRF4 (F) and IRF8 (H) expression. Data are shown as the mean ± SEM of five independent experiments. The significance was calculated using One-way ANOVA with Tukey’s post-test. n.s, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

Article Snippet: Thereafter, cells were collected, washed with PBS, fixed with 4% paraformaldehyde (PFA) for 40 min, and then permeabilized using the Foxp3 Staining Kit (eBioscience, San Diego, California, USA) for 35 min. After permeabilization, cells were incubated with anti-IRF4-PE (clone 3E4; cat# 12–9858-82), anti-IRF8APC (clone V3GYWCH; cat# 2093671), or with anti-Ezh2 (cat# 4905S, Cell Signaling Technology), and anti-USP7 primary Ab (cat# 4833, Cell Signaling Technology, Whitby, Ontario, Canada).

Techniques: Expressing, Purification, Cell Culture, Labeling, Flow Cytometry, Fluorescence

Journal: Cellular & molecular biology letters

Article Title: Role of USP7 in the regulation of tolerogenic dendritic cell function in type 1 diabetes.

doi: 10.1186/s11658-025-00727-5

Figure Lengend Snippet: Fig. 6 USP7 controls IRF4 and IRF8 expression by regulating Ezh2. A Crystal structure of overall protein– protein binding between USP7 and Ezh2. B Ribbon representation of the crystal structure of USP7:Ezh2 peptide complex with the USP7 peptide represented by blue color and Ezh2 peptide represented by red color (left). The interactions formed between USP7 and the Ezh2 peptide are shown as yellow dashed lines. The residues involved in the interactions are labeled (right) and presented in table form. C–G Splenic DCs purified from NOD and NOD.Stat5b-CA mice were incubated with either vehicle (0.1% DMSO) or with the Ezh2 inhibitor GSK343 (3 μM/mL) alone or together with the USP7 inhibitor P5091 (5 μM/mL) for 24 h. Thereafter, cells were washed and then stained with anti-CD11c, anti-Ezh2, anti-IRF4, and anti-IRF8 antibodies and analyzed by flow cytometry. C, D Histogram profile (left panel) and Mean fluorescence intensity (MFI) quantification (right panel) of the level of Ezh2 expression in CD11c+ DCs. Data are shown as the mean ± SEM of three independent experiments. The two-tailed unpaired Student’s t-test was used. **P < 0.01. E MFI values of Ezh2 in DCs treated with either vehicle or P5091 inhibitor. F, G MFI values of IRF4 and IRF8 in vehicle-treated DCs or DCs treated with Ezh2 inhibitor alone or together with P5091 inhibitor. Data are shown as the mean ± SEM of three independent experiments. One-way ANOVA followed by Tukey’s post hoc test was used. n.s, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

Article Snippet: Thereafter, cells were collected, washed with PBS, fixed with 4% paraformaldehyde (PFA) for 40 min, and then permeabilized using the Foxp3 Staining Kit (eBioscience, San Diego, California, USA) for 35 min. After permeabilization, cells were incubated with anti-IRF4-PE (clone 3E4; cat# 12–9858-82), anti-IRF8APC (clone V3GYWCH; cat# 2093671), or with anti-Ezh2 (cat# 4905S, Cell Signaling Technology), and anti-USP7 primary Ab (cat# 4833, Cell Signaling Technology, Whitby, Ontario, Canada).

Techniques: Expressing, Protein Binding, Labeling, Purification, Incubation, Staining, Flow Cytometry, Fluorescence, Two Tailed Test

Journal: Cellular & molecular biology letters

Article Title: Role of USP7 in the regulation of tolerogenic dendritic cell function in type 1 diabetes.

doi: 10.1186/s11658-025-00727-5

Figure Lengend Snippet: Fig. 7 Inhibition of USP7 in tolerogenic Stat5b-CA.DCs reduce Tregs and Th2 responses and promote IL-17-producing Th17 cell subset. Purified splenic DCs from NOD and NOD.Stat5b-CA mice were pre-incubated with (+ PD5091) or without (− PD509) USP7 inhibitor (5 µM/mL) for 24 h and then stimulated with LPS (1 µg/mL) for additional 24 h. Cells were then washed, and 6 × 106 cells were i.v injected into 8–10 weeks old NOD mice. After 7 days, spleen cells were harvested and analyzed for Tregs and Th1/Th2/Th17 cell subsets and their cytokine profiles by FACS. A–H Representative FACS profile and percentage of CD4+Foxp3+ Tregs (A, B), CD4+Gata-3+ Th2 (C-D), CD4+RORγt+ Th17 (E, F) and CD4+T-bet+ Th1 (G, H) cells. I–L Splenic cells of recipient NOD mice were stained with different T cell subsets and cytokine antibodies and analyzed by FACS. Percentages of Foxp3+IL-10+ (I), CD4+IL-4+ (J), CD4+IL-17+ (K), and CD4+IFN-γ+ (L) T cell subsets. In all experiments, 4 mice per group were used, and the results are expressed as mean ± SEM. The significance was calculated using One-way ANOVA with Tukey’s post-test. n.s., not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

Article Snippet: Thereafter, cells were collected, washed with PBS, fixed with 4% paraformaldehyde (PFA) for 40 min, and then permeabilized using the Foxp3 Staining Kit (eBioscience, San Diego, California, USA) for 35 min. After permeabilization, cells were incubated with anti-IRF4-PE (clone 3E4; cat# 12–9858-82), anti-IRF8APC (clone V3GYWCH; cat# 2093671), or with anti-Ezh2 (cat# 4905S, Cell Signaling Technology), and anti-USP7 primary Ab (cat# 4833, Cell Signaling Technology, Whitby, Ontario, Canada).

Techniques: Inhibition, Purification, Incubation, Injection, Staining

Journal: Cellular & molecular biology letters

Article Title: Role of USP7 in the regulation of tolerogenic dendritic cell function in type 1 diabetes.

doi: 10.1186/s11658-025-00727-5

Figure Lengend Snippet: Fig. 8 USP7 blockade halts the capacity of tolerogenic Stat5b-CA.DCs to protect NOD mice from diabetes. Purified splenic DC of NOD and NOD.Stat5b-CA mice were treated or not with USP7 inhibitor P5091 (5 µM/mL, for 24 h), stimulated with LPS for another 24 h, and intravenously injected into 3–4 weeks old female NOD mice (7 mice per group, 6 × 106 cells/mouse). Recipient NOD mice were followed for diabetes development until 32 weeks of age. For comparison of diabetes incidence between different groups, the Gehan–Breslow–Wilcoxon test was performed to calculate the significant difference. n.s., not significant; ****P < 0.0001

Article Snippet: Thereafter, cells were collected, washed with PBS, fixed with 4% paraformaldehyde (PFA) for 40 min, and then permeabilized using the Foxp3 Staining Kit (eBioscience, San Diego, California, USA) for 35 min. After permeabilization, cells were incubated with anti-IRF4-PE (clone 3E4; cat# 12–9858-82), anti-IRF8APC (clone V3GYWCH; cat# 2093671), or with anti-Ezh2 (cat# 4905S, Cell Signaling Technology), and anti-USP7 primary Ab (cat# 4833, Cell Signaling Technology, Whitby, Ontario, Canada).

Techniques: Purification, Injection, Comparison