Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: PCV2 infection led to translocation of HMGB1 from nuclei to cytoplasmic compartments. PK-15 cells and porcine monocytic cells (3D4/31) were infected for 36 h with PCV2 (MOI = 1) or mock infected as a control. (A) Confocal imaging of HMGB1 distribution in PCV2-infected cells immunostained with anti-HMGB1 (green) and anti-Cap (red) antibodies. Nuclei were labeled with DAPI (blue). Representative micrographic images are shown. (B) Immunoblotting of PCV2 Cap and HMGB1 in nuclear and cytoplasmic extracts from PCV2- or mock-infected PK-15 cells. Histone H3 and GAPDH were used as internal controls for nuclear and cytoplasmic fractions, respectively. (C) The intensity of protein bands was quantified densitometrically using Gel-Pro Analyzer. Ratios of nuclear or cytoplasmic HMGB1 to Histone H3 or GAPDH were quantified, respectively. (D and E) Quantification of hmgb1 mRNA by qPCR in PK-15 and 3D4/31 cells infected with PCV2 for different times using total RNA extracts from the cells. (F and G) Immunoblotting of HMGB1 and PCV2 Cap in the lysates of PK-15 and 3D4/31 cells infected with PCV2 for different times. β-Actin was used as a loading control. The data in panels A, B, F, and G are representative of three independent experiments. Bar charts in panels C, D, and E show means ± SDs from three independent experiments. ns, not significant; *, P < 0.05; **, P < 0.01.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Infection, Translocation Assay, Imaging, Labeling, Western Blot

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: Overexpression of HMGB1 inhibited PCV2 replication. PK-15 cells were transfected with recombinant plasmid expressing HMGB1 (pHMGB1) or control plasmid (pFlag) for 24 h and then infected with PCV2 (MOI = 1) for 36 h. (A) Effect of HMGB1 overexpression on PCV2 Cap expression as shown by immunoblotting using protein samples from the whole-cell lysates. β-Actin was used as a loading control. The gel shown is representative of three independent experiments. (B) The ratios of band intensity of HMGB1 or PCV2 Cap to β-actin (as shown in panel A). (C) Effect of HMGB1 overexpression on PCV2 orf2 (encoding Cap) transcription measured by qPCR using total RNA extracted from the whole-cell lysates. (D) PCV2 replication in cells overexpressing HMGB1 as assessed by indirect immunofluorescence. Percentages of PCV2-infected cells were calculated as described in the legend for Fig. 2. Relative percentages of PCV2-infected cells in the HMGB1 overexpressing cells are shown with nontransfected but PCV2-infected cells set at 100%. (E) PCV2 genomic DNA copies in cells overexpressing HMGB1 quantified by qPCR using total DNA extracts from whole-cell lysates. Bar charts in panels B, C, D, and E show means ± SDs from three independent experiments. ns, not significant; **, P < 0.01; ***, P < 0.001.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Over Expression, Transfection, Recombinant, Plasmid Preparation, Expressing, Infection, Western Blot, Immunofluorescence

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: Downregulation of HMGB1 promoted PCV2 replication. PK-15 cells were transfected with hmgb1-specific RNA interference (RNAi) plasmid (sh-HMGB1) or control RNAi plasmid (sh-NC) for 24 h and then infected with PCV2 (MOI= 1) for 36 h. (A) Effect of hmgb1 knockdown on PCV2 Cap expression (β-actin used as a loading control) as shown by immunoblotting using protein samples from the whole-cell lysates. The gel shown is representative of three independent experiments. (B) The ratios of band intensity of HMGB1 or PCV2 Cap to β-actin (as shown in panel A). (C) Effect of hmgb1 knockdown on PCV2 orf2 (encoding Cap) transcription examined by qPCR using total RNA extracted from the whole-cell lysates. (D) PCV2 replication in hmgb1-silenced cells as assessed by indirect immunofluorescence. Representative fluorescence images are shown (top). Percentage of PCV2-infected cells was calculated by dividing the number of PCV2-infected cells by the total cell number in each group (n = 2 images for each experiment per group) that were counted using ImageJ software. Relative percentages of PCV2-infected cells in the hmgb1-silenced cells are shown with nontransfected but PCV2-infected cells set at 100% (bottom). (E) Effect of hmgb1 silencing on PCV2 genomic DNA copies measured by qPCR using total DNA extracts from whole-cell lysates. Bar charts in panels B, C, D, and E show means ± SDs from three independent experiments. ns, not significant; **, P < 0.01.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Transfection, Plasmid Preparation, Infection, Expressing, Western Blot, Immunofluorescence, Fluorescence, Software

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: Nuclear HMGB1 repressed PCV2 replication. PK-15 cells were transfected with recombinant plasmid expressing HMGB1 (pHMGB1) or control plasmid (pFlag) for 24 h and then infected with PCV2 (MOI = 1) for 36 h. Nuclear and cytoplasmic extracts were prepared for immunoblotting as described in the legend for Fig. 1 Immunoblotting of HMGB1 and PCV2 Cap in the nuclear (A) and cytoplasmic (B) fractions. Histone H3 and GAPDH were used as internal controls for nuclear and cytoplasmic extracts, respectively. Representative images from three independent experiments are shown. The ratios of band intensities of HMGB1 or PCV2 Cap to those of histone H3 (as shown in panel A) in the nuclear fraction (C) or to GAPDH (as shown in panel B) in the cytoplasmic fraction (D). (E) Effect of HMGB1 overexpression on PCV2 orf2 transcription in the nuclei examined by qPCR using total RNA extracted from the nuclear fractions. Results were normalized to histone H3 mRNA in the same samples. (F) PCV2 genomic DNA replication in the nuclei of HMGB1-overexpressing cells quantified by qPCR using total DNA extracted from nuclear fractions. Bar charts in panels C to F show means ± SDs from three independent experiments. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Transfection, Recombinant, Plasmid Preparation, Expressing, Infection, Western Blot, Over Expression

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: Ethyl pyruvate inhibited nucleocytoplasmic translocation of HMGB1 in PCV2-infected cells. PK-15 cells were mock infected or infected with PCV2 (MOI = 1) with or without ethyl pyruvate (EP; 7.5 mM) treatment. The cell samples were harvested at 36 hpi. (A) Confocal microscopic images show inhibition of nuclear HMGB1 migration into the cytosol by EP. Cells were immunostained for HMGB1 (green) and PCV2 Cap (red), with nuclei stained with DAPI (blue). Bars, 10 μm. (B) Immunoblotting of HMGB1 and PCV2 Cap in the nuclear and cytoplasmic fractions of PCV2-infected and EP-treated cells. Histone H3 and GAPDH were used as internal controls for nuclear and cytoplasmic extracts, respectively. Representative images from three independent experiments are shown. The ratios of band intensities of HMGB1 or PCV2 Cap to those of histone H3 (as shown in panel B, left) in the nuclear fraction (C) or to GAPDH (as shown in panel B, right) in the cytoplasmic fraction (D). (E) Effect of EP on PCV2 genomic DNA replication by qPCR using DNA extracted from nuclei of PCV2-infected cells treated with 7.5 mM EP. Bar charts in panels C, D, and E show means ± SDs from three independent experiments. ns, not significant; **, P < 0.01.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Translocation Assay, Infection, Inhibition, Migration, Staining, Western Blot

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: N-Acetylcysteine inhibited PCV2-induced HMGB1 translocation from nuclei to cytosol and repressed PCV2 replication. PK-15 cells were mock infected or infected with PCV2 (MOI = 1) for 12 h and then treated with 10 mM N-acetylcysteine (NAC). The cell samples were harvested at 36 hpi. (A) Confocal imaging of HMGB1 distribution in PCV2-infected and NAC-treated cells after the cells were fixed and immunostained for HMGB1 (green) and Cap (red). Nuclei were stained with DAPI (blue). Bars, 10 μm. (B) Blotting of HMGB1 and PCV2 Cap in the nuclear and cytoplasmic extracts of PCV2-infected cells with or without NAC treatment. Histone H3 and GAPDH were used as internal controls for the nuclear and cytoplasmic fractions, respectively. The figure is representative of three independent experiments. The ratios of band intensities of HMGB1 or PCV2 Cap to histone H3 (as shown in panel B, left) in the nuclear fraction (C) or to GAPDH (as shown in panel B, right) in the cytoplasmic fraction (D). (E) Effect of NAC on PCV2 genomic DNA replication by qPCR using DNA extracted from lysates of PCV2-infected cells treated with NAC. (F) Cytosolic ROS levels in PCV2-infected cells with or without treatment by NAC or ethyl pyruvate (EP) as measured by flow cytometry after probing with DCFH-DA. Bar charts in panels C, D, E, and F show means ± SDs from three independent experiments. *, P < 0.05; **, P < 0.01.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Translocation Assay, Infection, Imaging, Staining, Flow Cytometry

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: Ethyl pyruvate was inhibitory to PCV2 infection. PK-15 cells were mock infected or infected with PCV2 (MOI = 1) with or without ethyl pyruvate (EP; 7.5 mM) treatment. The cell samples were harvested at 36 hpi. (A) Effect of EP on PCV2 replication in PK-15 cells by immunofluorescence. Percentages of PCV2-infected cells were calculated as described in the legend for Fig. 2. Relative percentages of PCV2-infected cells in the EP-treated cells are shown with untreated but PCV2-infected cells set at 100%. (B) Immunoblotting of HMGB1 and PCV2 Cap in whole-cell lysates with β-actin used as a loading control. (C) The ratios of band intensities of HMGB1 or PCV2 Cap to β-actin (as shown in panel B). Bar charts in panels A and C show means ± SDs from three independent experiments. ns, not significant; **, P < 0.01.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Infection, Immunofluorescence, Western Blot

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: The B box domain of HMGB1 was involved in inhibition of PCV2 replication. (A) Schematic illustration of the full-length and truncated forms of porcine HMGB1 according to its human homolog. All truncated versions, A box, AB box, and B box plus C terminus (B boxCT), were flag tagged. The numbers indicate positions of amino acids. Arrows with C followed by numbers represent key cysteine residues. NLS, nuclear localization signal. PK-15 cells were transfected with recombinant plasmids expressing flag-tagged or full-length HMGB1 for 24 h and then infected with PCV2 (MOI = 1) for 36 h. (B) Numbers of PCV2-infected cells examined by immunofluorescence using anti-Cap monoclonal antibody as the probe (top). Expression of PCV2 Cap and different forms of HMGB1 as assessed by immunoblotting using the whole-cell lysates harvested at 36 hpi and antibodies against Flag, Cap, and HMGB1 (bottom). β-Actin was used as a loading control. The panel B images are representative of three individual experiments. (C) The ratios of band intensities of PCV2 Cap to those of β-actin (as shown at the bottom panel of B). (D) Effect of different HMGB1 truncations on PCV2 DNA replication estimated by qPCR using total DNA extracted from the whole-cell lysate. Bar charts in panels C and D show means ± SDs from three independent experiments. ns, not significant; **, P < 0.01.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Inhibition, Transfection, Recombinant, Expressing, Infection, Immunofluorescence, Western Blot

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: HMGB1 bound to the Ori region of the PCV2 genome. (A) Binding of porcine HMGB1 to PCV2 DNA using the gel shift assay. PCV2 DNA (500 ng) and various concentrations (0 to 5 μg) of purified His-tagged recombinant HMGB1 were mixed in binding buffer. The DNA-protein mixtures were subjected to 0.8% agarose gel electrophoresis to visualize changes of the DNA motility. (B) Binding of HMGB1 to a specific region of PCV2 DNA: full-length and different fragments of PCV2 genome (orf1, orf2, and Ori) were incubated with recombinant HMGB1 protein to identify the region of PCV2 genome involved in HMGB1 binding. (C) To confirm the Ori region is required for HMGB1 binding, the Ori fragment was combined with orf1 or orf2 (Ori-orf1 or Ori-orf2) that were then compared with orf1 or orf2 alone by the gel shift assay. (D) Immunoprecipitation of purified HMGB1 protein (500 μg) and PCV2 DNA (500 ng) mixture by anti-HMGB1 antibody (rabbit IgG as control) and protein A/G agarose. The precipitates were probed with anti-His and anti-HMGB1 antibodies by immunoblotting. (E) Quantification of PCV2 genomic Ori copies by qPCR in DNA extracts from PCV2 DNA-HMGB1 precipitates (shown in panel D) after DNase pretreatment. (F) Blotting of HMGB1 in immunoprecipitates of whole-cell lysates (WCL) of the PK-15 cells infected with PCV2 (36 h) by anti-HMGB1 (rabbit IgG as control) and protein A/G agarose. (G) Quantification of PCV2 genomic Ori copies by qPCR in DNA extracts from immunoprecipitates of whole-cell lysates (shown in panel F) after DNase pretreatment. (H) Blotting of HMGB1 in immunoprecipitates of nuclear extracts of the PK-15 cells infected with PCV2. (I) Quantification of PCV2 genomic Ori copies in the precipitates shown in panel H. Bar charts in panels E, G, and I show means ± SDs from three independent experiments.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Binding Assay, Electrophoretic Mobility Shift Assay, Purification, Recombinant, Agarose Gel Electrophoresis, Incubation, Immunoprecipitation, Western Blot, Infection

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: Effect of hydrogen peroxide treatment on subcellular localization of HMGB1 and PCV2 replication. (A) H2O2 treatment promoted nucleocytoplasmic translocation of HMGB1. PK-15 cells were treated with or without N-acetylcysteine (NAC; 10 mM) before adding 50 μM H2O2. Cells were fixed and immunostained with anti-HMGB1 (green) for confocal microscopy. Nuclei were labeled with DAPI (blue). (B) Immunoblotting of HMGB1 in the nuclear and cytoplasmic fractions of PK-15 cells treated with H2O2 and NAC. (C) Confocal imaging of PK-15 cells infected by PCV2 with or without 50 μM H2O2 treatment after immunostaining with anti-HMGB1 (green) and anti-Cap (red) antibodies. (D) Blotting of HMGB1 and PCV2 Cap in the nuclear and cytoplasmic extracts of PCV2-infected cells with or without H2O2 treatment. Histone H3 and GAPDH were used as internal controls for the nuclear and cytoplasmic fractions, respectively. (E) Percentages of PCV2-infected cells were calculated from immunofluorescence images as described in the legend for Fig. 2. Relative percentages of PCV2-infected cells in the H2O2-treated cells are shown with nontreated but PCV2-infected cells set at 100%. Bar chart in panel E shows means ± SDs from three independent experiments. **, P < 0.01.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Translocation Assay, Confocal Microscopy, Labeling, Western Blot, Imaging, Infection, Immunostaining, Immunofluorescence

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: Schematic illustration of the interaction between PCV2 and HMGB1 in infected cells. HMGB1 in the nucleus restricts PCV2 replication by binding to the Ori region of the PCV2 genome. PCV2 infection causes increased generation of cellular ROS. Increased ROS promotes nucleocytoplasmic translocation of HMGB1 and lessens sequestration of the viral DNA by HMGB1 in the nucleus, thus enhancing PCV2 replication. N-Acetylcysteine (and probably ethyl pyruvate as well) scavenges PCV2-induced ROS and thus increases retention of HMGB1 in the nucleus, leading to sequestration of viral DNA and reduced PCV2 replication.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Infection, Binding Assay, Translocation Assay

Journal:

Article Title: Nuclear Heat Shock Protein 72 as a Negative Regulator of Oxidative Stress (Hydrogen Peroxide)-Induced HMGB1 Cytoplasmic Translocation and Release 1

doi:

Figure Lengend Snippet: A mild heat shock (HS) pretreatment attenuates oxidative stress (H2O2)-induced HMGB1 release in macrophages cultures. A, Effects of mild or severe heat shock on HMGB1 release. RAW 264.7 cells were subjected to a brief heat shock (1 h) at 42.5°C (mild heat shock), 43.5°C or higher (severe heat shock), after which was returned to 37°C and incubated for additional 12 h. HMGB1 in the culture medium were detected by Western blotting analysis (top). In parallel, the cell viability was determined by LDH release assay (bottom). Ctrl, control cells. Blot is representative of three experiments with similar results. Values are mean ± SEM (n = 3) of three experiments in duplicate. B, Mild HS pretreatment induced Hsp expression. RAW 264.7 cells were subjected to heat shock (42.5°C, 1 h), and cellular levels of Hsp90 and Hsp72 were detected by Western blotting analysis at the indicated time points after heat shock. In parallel, levels of HMGB1 in the culture medium were determined by Western blotting analysis. Tubulin was used as a loading control. Values are representative of three independent experiments with similar results. C, Effects of oxidative stress on Hsp72 expression. RAW 264.7 cells were stimulated with H2O2 at nontoxic (0.125 mM), or low-toxic (0.25 mM) concentrations, and cellular Hsp72 were detected at 12 h poststimulation by Western blotting analysis. GAPDH was used as a loading control. Values are representative of three independent experiments with similar results. D, Mild heat shock pretreatment inhibited H2O2-induced HMGB1 release. After mild heat shock (42.5°C, 1 h), cells were allowed to recover for 12 h at 37°C and then stimulated for 12 h with H2O2 at nontoxic (0.125 mM) or low-toxic (0.25 mM) concentrations. Levels of HMGB1 in the culture medium were determined by Western blotting and expressed (in arbitrary units; AU) as mean ± SEM of three experiments in duplicate. In parallel, the cell viability was determined by MTT assay, and expressed as mean ± SEM (n = 4) of three experiments in duplicate. *, p < 0.05.

Article Snippet: Cover slips were saturated with PBS containing 2% BSA for 1 h at room temperature and processed for immunofluorescence with rabbit anti-HMGB1 polyclonal Ab (BD Biosciences) or mouse anti-Hsp72 mAb (Stressgen) followed by Cy3-conjugated sheep anti-rabbit Ig (Sigma-Aldrich) or FITC-conjugated sheep anti-mouse Ig (Boster Biotech), respectively.

Techniques: Incubation, Western Blot, Lactate Dehydrogenase Assay, Control, Expressing, MTT Assay

Journal:

Article Title: Nuclear Heat Shock Protein 72 as a Negative Regulator of Oxidative Stress (Hydrogen Peroxide)-Induced HMGB1 Cytoplasmic Translocation and Release 1

doi:

Figure Lengend Snippet: Overexpression of Hsp72 renders macrophages resistant to H2O2-induced HMGB1 release. A, Western blotting analysis of Hsp72 levels in RAW 264.7 cells transfected with empty plasmid (lane 1), Hsp72 expression construct (lane 2) or challenged with mild heat shock (HS, 42.5°C, 1 h). Tubulin was used as a loading control. B, Visualization of fluorescent Hsp72 protein in RAW 264.7 cells transfected with Hsp72 expression construct (top), or empty plasmid (bottom). Nuclei were visualized by Hoechst staining. C, Western blotting analysis of H2O2-induced HMGB1 release in RAW 264.7 cells transfected with empty plasmid, or Hsp72 expression construct. RAW 264.7 cells were stimulated with H2O2 (0.125 and 0.25 mM) for 12 h, and HMGB1 levels in the culture medium were determined, and expressed as mean ± SEM of three experiments in duplicate. *, p < 0.05.

Article Snippet: Cover slips were saturated with PBS containing 2% BSA for 1 h at room temperature and processed for immunofluorescence with rabbit anti-HMGB1 polyclonal Ab (BD Biosciences) or mouse anti-Hsp72 mAb (Stressgen) followed by Cy3-conjugated sheep anti-rabbit Ig (Sigma-Aldrich) or FITC-conjugated sheep anti-mouse Ig (Boster Biotech), respectively.

Techniques: Over Expression, Western Blot, Transfection, Plasmid Preparation, Expressing, Construct, Control, Staining

Journal:

Article Title: Nuclear Heat Shock Protein 72 as a Negative Regulator of Oxidative Stress (Hydrogen Peroxide)-Induced HMGB1 Cytoplasmic Translocation and Release 1

doi:

Figure Lengend Snippet: Overexpression of Hsp72 attenuates H2O2-induced HMGB1 cytoplasmic translocation in macrophage cultures. A and B, H2O2 induces transient nuclear translocation of Hsp72 in macrophages cultures. pcDNA3.1-Hsp72-transfected RAW 264.7 cells were stimulated with H2O2 (0.125 mM) for various period of time and examined for Hsp72 subcellular localization by immunocytochemistry (A) or cell fractionation/Western blot (B). Green, Hsp72; blue, nuclei. Original magnification, ×400. A nuclear protein, proliferating cell nuclear Ag, was used as a loading control. C, Heat shock induces Hsp72 expression and nuclear translocation in macrophages cultures. RAW 264.7 cells subject to mild heat shock (42.5°C, 1 h), and nuclear Hsp72 content was determined by Western blotting. D, Overexpression of Hsp72 attenuates H2O2-induced HMGB1 cytoplasmic translocation. RAW 264.7 cells transfected with empty pcDNA3.1 plasmid, or pcDNA3.1-Hsp72 construct were stimulated with H2O2 (0.125 mM) for 12 h, and the subcellular localization of HMGB1 was determined by immunocytochemical analysis (D, left). Relative fluorescence intensity of HMGB1 in the nuclear (N) and cytoplasmic (C) regions of multiple representative cells was assayed using the ImageProPlus software (D, right). Image is representative of three experiments with similar results. Red, HMGB1; blue, nuclei. Original magnification, ×1000. Values are means ± SEM (n = 50) of three experiments in duplicate. *, p < 0.05.

Article Snippet: Cover slips were saturated with PBS containing 2% BSA for 1 h at room temperature and processed for immunofluorescence with rabbit anti-HMGB1 polyclonal Ab (BD Biosciences) or mouse anti-Hsp72 mAb (Stressgen) followed by Cy3-conjugated sheep anti-rabbit Ig (Sigma-Aldrich) or FITC-conjugated sheep anti-mouse Ig (Boster Biotech), respectively.

Techniques: Over Expression, Translocation Assay, Transfection, Immunocytochemistry, Cell Fractionation, Western Blot, Control, Expressing, Plasmid Preparation, Construct, Fluorescence, Software

Journal:

Article Title: Nuclear Heat Shock Protein 72 as a Negative Regulator of Oxidative Stress (Hydrogen Peroxide)-Induced HMGB1 Cytoplasmic Translocation and Release 1

doi:

Figure Lengend Snippet: Hsp72 and HMGB1 are coimmunoprecipitated after oxidative stress or heat shock. A, Coimmunoprecipitation of Hsp72 and HMGB1 from lysate of RAW 264.7 cells. RAW 264.7 cells transfected with pcDNA3.1-Hsp72 construct were stimulated with H2O2 (0.125 mM) for 3 h, and whole-cell lysate was immunoprecipitated with various Abs: nonspecific rabbit serum (Sr), nonspecific mouse serum (Sm), or Abs specific for Hsp72 or HMGB1, respectively. In parallel experiment, RAW 264.7 cells were subjected to mild heat shock (42.5°C for 1 h) and returned to 37°C for 6 h, and whole-cell lysate was immunoprecipitated with HMGB1- or Hsp72-specific Abs, respectively. The precipitated complexes were separately immunoblotted with Hsp72- or HMGB1-specific Abs. IP, immunoprecipitation; IB, Immunoblotting. Hsp72-transfected cell lysate (L) was used as a positive control. Blot is representative of three experiments with similar results. B, Coimmunoprecipitation of Hsp72 and HMGB1 from lysate of human leukemia K562 cells. K562 cells were subjected to oxidative stress (by stimulating withH2O2, 0.125 mM, for 12 h) or mild heat shock (HS, 42.5°C, 1 h, and then returned to 37°C for 6 h), and whole-cell lysate was immunoprecipitated with HMGB1-specific Abs. The precipitated complexes were then sequentially blotted with Hsp72- or HMGB1-specific Abs, respectively. IP, immunoprecipitation; IB, immunoblotting; Ctrl, control cells. Blot is representative of two experiments with similar results. C, Coimmunoprecipitation of Hsp72 and HMGB1 from nuclear and cytoplasmic fraction of RAW 264.7 cells transfected with pcDNA3.1-Hsp72. Cells were subjected to oxidative stress (H2O2, 0.125 mM for 3 h; H), or mild heat shock (42.5°C for 1 h, then returned to 37°C for 6 h; HS), and nuclear or cytoplasmic proteins were immunoprecipitated with Hsp72-or HMGB1-specific Abs. The precipitated complexes were then blotted with Hsp72- or HMGB1-specific Abs. Hsp72-transfected cell lysates was used as a positive control. C, control cells. Blot is representative of two experiments with similar results. D, Stoichiometry analysis of Hsp72-bound HMGB1 in nuclear fractions. RAW 264.7 cells transfected with pcDNA3.1-Hsp72 were stimulated with H2O2 (0.125 mM) for 3 h, and levels of HMGB1 in the whole-cell extracts before (1, set at 100%) or after (2) immunoprecipitation with excessive amount of Hsp72-specific Ab was determined by Western blotting. Blot is representative of three experiments with similar results.

Article Snippet: Cover slips were saturated with PBS containing 2% BSA for 1 h at room temperature and processed for immunofluorescence with rabbit anti-HMGB1 polyclonal Ab (BD Biosciences) or mouse anti-Hsp72 mAb (Stressgen) followed by Cy3-conjugated sheep anti-rabbit Ig (Sigma-Aldrich) or FITC-conjugated sheep anti-mouse Ig (Boster Biotech), respectively.

Techniques: Transfection, Construct, Immunoprecipitation, Western Blot, Positive Control, Control

Journal:

Article Title: Nuclear Heat Shock Protein 72 as a Negative Regulator of Oxidative Stress (Hydrogen Peroxide)-Induced HMGB1 Cytoplasmic Translocation and Release 1

doi:

Figure Lengend Snippet: An herb-derived antioxidant, quercetin, attenuates H2O2-induced Hsp72 nuclear translocation, and Hsp72-HMGB1 interaction in macrophage cultures. RAW 264.7 cells transfected with pcDNA3.1-Hsp72 construct were pretreated with quercetin (50 μM) for 4 h and subsequently stimulated with H2O2 (0.125 mM) for 3 h. Subcellular localization of Hsp72 in cells was determined by immunocytochemistry (A) or Western blot (B). Results are representative of three experiments with similar results. Green, Hsp72; blue, nuclei. Original magnification, ×400). Ctrl, control cells; Q, + quercetin; H, + H2O2; QH, + quercetin + H2O2. In parallel experiments, whole-cell lysate was immunoprecipitated with Hsp72-specific Abs, and the precipitated complexes were immunoblotted with Hsp72- or HMGB1-specific Abs, respectively (C). IP, immunoprecipitation; IB, immunoblotting. Blot is representative of three experiments with similar results.

Article Snippet: Cover slips were saturated with PBS containing 2% BSA for 1 h at room temperature and processed for immunofluorescence with rabbit anti-HMGB1 polyclonal Ab (BD Biosciences) or mouse anti-Hsp72 mAb (Stressgen) followed by Cy3-conjugated sheep anti-rabbit Ig (Sigma-Aldrich) or FITC-conjugated sheep anti-mouse Ig (Boster Biotech), respectively.

Techniques: Derivative Assay, Translocation Assay, Transfection, Construct, Immunocytochemistry, Western Blot, Control, Immunoprecipitation

Journal:

Article Title: Nuclear Heat Shock Protein 72 as a Negative Regulator of Oxidative Stress (Hydrogen Peroxide)-Induced HMGB1 Cytoplasmic Translocation and Release 1

doi:

Figure Lengend Snippet: Identification of functional domains of Hsp72 for interacting with nuclear HMGB1. A, Schematic diagram of Hsp72 and two mutants lacking the NLS or PBD. B, Detection of HMGB1 and Myc-tagged Hsp72, Hsp72-ΔNLS, or Hsp72-ΔPBD by Western blotting. RAW 264.7 cells were transiently transfected with pcDNA3.1-Hsp72, pcDNA3.1-Hsp72-ΔNLS, or pcDNA3.1-Hsp72-ΔPBD and stimulated with H2O2 (0.125 mM) for 3 h; nuclear extract was assayed for HMGB1 and Myc-tagged proteins by Western blotting (Immunoblotting; IB) analysis. Blots are representative of two independent experiments with similar results. C, Coimmunoprecipitation of HMGB1 with Myc-tagged Hsp72, Hsp72-ΔNLS, or Hsp72-ΔPBD. In parallel experiments, nuclear extracts were immunoprecipitated (IP) with HMGB1-specific Abs, and the precipitated complexes were then assayed for levels of HMGB1 or Myc-tagged proteins by Western blotting. Blots are representative of two independent experiments with similar results. D, Western blotting of H2O2-induced HMGB1 release (D, left) and translocation (D, right) in RAW 264.7 cells transfected with empty plasmid, pcDNA3.1-Hsp72, pcDNA3.1-Hsp72-ΔNLS, or pcDNA3.1-Hsp72-ΔPBD. Tubulin was used as a loading control. Blots are representative of three independent experiments with similar results.

Article Snippet: Cover slips were saturated with PBS containing 2% BSA for 1 h at room temperature and processed for immunofluorescence with rabbit anti-HMGB1 polyclonal Ab (BD Biosciences) or mouse anti-Hsp72 mAb (Stressgen) followed by Cy3-conjugated sheep anti-rabbit Ig (Sigma-Aldrich) or FITC-conjugated sheep anti-mouse Ig (Boster Biotech), respectively.

Techniques: Functional Assay, Western Blot, Transfection, Immunoprecipitation, Translocation Assay, Plasmid Preparation, Control

Journal:

Article Title: Nuclear Heat Shock Protein 72 as a Negative Regulator of Oxidative Stress (Hydrogen Peroxide)-Induced HMGB1 Cytoplasmic Translocation and Release 1

doi:

Figure Lengend Snippet: Hypothetical role of Hsp72 in the regulation of oxidative stress-induced HMGB1 cytoplasmic translocation and release. In response to stresses (e.g., heat shock), Hsp72 is produced to maintain a pool of Hsp72 in the cytoplasm. Upon stimulation with secondary oxidative stress (e.g., H2O2), Hsp72 is translocated into the nucleus, where it directly, or indirectly, interacts with various nuclear proteins (such as HMGB1 and histone deacetylase 1 (HDAC1). The intranuclear Hsp72-HMGB1 consequently prevents HMGB1 cytoplasmic translocation and subsequent release via the secretory lysosome pathway.

Article Snippet: Cover slips were saturated with PBS containing 2% BSA for 1 h at room temperature and processed for immunofluorescence with rabbit anti-HMGB1 polyclonal Ab (BD Biosciences) or mouse anti-Hsp72 mAb (Stressgen) followed by Cy3-conjugated sheep anti-rabbit Ig (Sigma-Aldrich) or FITC-conjugated sheep anti-mouse Ig (Boster Biotech), respectively.

Techniques: Translocation Assay, Produced, Histone Deacetylase Assay

Journal: Gene therapy

Article Title: Measles virus causes immunogenic cell death in human melanoma

doi: 10.1038/gt.2011.205

Figure Lengend Snippet: The inflammatory response associated with MV infection. A; Cytokine/chemokine release. Cell-free supernatants were collected 48 hours after infection with MV and cytokine levels determined by ELISA. Data shown are representative of three independent experiments. B; HMGB1 release. Melanoma cells were treated with MV at MOI from 0.01 to 5. 48hours after infection cell-free supernatant was collected then analysed by western blot for HMGB1. Lanes with protein markers (M) and untreated controls (C) are indicated. Data shown are representative of two separate experiments.

Article Snippet: The supernatants were diluted 1:1 with Laemmli buffer prior to loading 20μL of diluted supernatant on a 10% SDS page gel using standard protocols; staining for HMGB1 was performed using a monoclonal mouse anti-human HMGB1 antibody (R&D systems, Abingdon UK) used at 1μg/mL in a 1:1 mix of Odyssey blocking buffer (LiCor Biotechnology, Cambridge UK) and PBS/0.1% Tween.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: HMGB1 in severe soft tissue infections caused by Streptococcus pyogenes

doi: 10.3389/fcimb.2014.00004

Figure Lengend Snippet: HMGB1 at the local site of infection . Snap-frozen tissue biopsies ( n = 25) from patients with severe soft tissue infections caused by S. pyogenes were immunohistochemically stained for S. pyogenes , macrophages identified by CD68-positivity, neutrophils identified by neutrophil elastase positivity, and HMGB1. A control staining where the primary antibody was omitted was also performed (control). (A) Representative immunohistochemically stained biopsies taken from the site of infection at day 1 from two patients (patient 8271, top panel, and patient 8611, bottom panel) (Table ). Positive stainings for cell markers and for HMGB1 are indicated by a brown color, and all cellular nuclei are stained blue by haematoxylin. Bars indicate 50 μm. (B) Staining's for HMGB1 revealed both a diffuse (top picture, indicated by white stars) and a distinct intracellular staining (bottom picture, indicated by the black arrow). Bars indicate 50 μm. (C) Image analysis values for S. pyogenes , macrophages (CD68), neutrophils (NE), and HMGB1 in the different biopsies were obtained by ACIA; for details see Materials and Methods. The horizontal lines denote median.

Article Snippet: HMGB1 was initially identified using a polyclonal anti-HMGB1 from BD Biosciences Pharmingen (556528, BD Diagnostics, San Diego, CA) or anti-human HMGB1 (ab18256, Abcam, Cambridge, UK).

Techniques: Infection, Staining

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: HMGB1 in severe soft tissue infections caused by Streptococcus pyogenes

doi: 10.3389/fcimb.2014.00004

Figure Lengend Snippet: HMGB1 correlates with severity of infection . Snap-frozen tissue biopsies ( n = 11) from patients with erysipelas were immunohistochemically stained for S. pyogenes , neutrophils identified by neutrophil elastase positivity and HMGB1. (A) Correlation between neutrophil infiltration in relation to HMGB1 ACIA value (for details see Materials and Methods) in erysipelas biopsies. Significant correlation, as determined by Pearson's correlation test, is indicated by p - and r -values. (B) Image analysis data of HMGB1 in tissue from patients with erysipelas were compared with biopsies from patients with severe streptococcal soft tissue infection of clinical grade 1 and 2 (for details see Materials and Methods). Statistically significant differences were determined by 1way ANOVA, Kruskal-Wallis; p = 0.0023. (C) Representative immunohistochemical pictures of erysipelas tissue biopsies collected at the inflamed center, 5 cm outside the lesion and from the healthy leg. Bars indicate 50 μm. (D) Acquired computerized image analysis values of the whole tissue biopsy.

Article Snippet: HMGB1 was initially identified using a polyclonal anti-HMGB1 from BD Biosciences Pharmingen (556528, BD Diagnostics, San Diego, CA) or anti-human HMGB1 (ab18256, Abcam, Cambridge, UK).

Techniques: Infection, Staining, Immunohistochemical staining

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: HMGB1 in severe soft tissue infections caused by Streptococcus pyogenes

doi: 10.3389/fcimb.2014.00004

Figure Lengend Snippet: Elevated HMGB1 in circulation in patients with STSS . HMGB1 and IL-8 levels in plasma collected on the day of inclusion were measured with ELISA and Luminex respectively, for details see Materials and Methods. Shown in (A) is systemic HMGB1 in STSS patients ( n = 19) and healthy controls ( n = 14). Statistically significant differences were determined by two-tailed t -test; p = 0.0001. Shown in (B) is systemic HMGB1 and IL-8 in patients with STSS ( n = 19 patients). Significant correlation, as determined by Pearson's correlation test, indicated by p - and r - values.

Article Snippet: HMGB1 was initially identified using a polyclonal anti-HMGB1 from BD Biosciences Pharmingen (556528, BD Diagnostics, San Diego, CA) or anti-human HMGB1 (ab18256, Abcam, Cambridge, UK).

Techniques: Enzyme-linked Immunosorbent Assay, Luminex, Two Tailed Test

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: HMGB1 in severe soft tissue infections caused by Streptococcus pyogenes

doi: 10.3389/fcimb.2014.00004

Figure Lengend Snippet: Cellular source of HMGB1 at the site of infection and chemotactic effect of HMGB1 on neutrophils . The images depicts a representative cryosections of tissue biopsies from patients with erysipelas, immunofluorescently stained for (A,B,D) HMGB1 (green) in combination with (A) macrophages (red) identified by CD68-positivity, (B) mast cells tryptase, or (D) neutrophils (red) identified by neutrophil elastase. Note the granular staining of HMGB1 (A,D) . Cell nuclei are stained blue with DAPI. Bars indicate 10 μm (A) and 20 μm (B,D) . (C) Human monocyte-derived macrophages were isolated from healthy blood donors and infected with S. pyogenes or E. coli at an MOI of 1–11 for 4 h. Supernatants were collected and analyzed for HMGB1 using an ELISA. Uninfected macrophages were used as control. The graph shows the results obtained from two different donors. (E) Western blot of recombinant HMGB1 and protein extracts from patient tissue biopsies (patient 5626 and 8612). Proteins were separated using a 12% NuPAGE Bis-Tris gel and visualized using a monoclonal anti-HMGB1 antibody. (F) The chemotactic effect of HMGB-1 on primary neutrophils was measured using a transwell assay system where neutrophils were left to migrate against 50 and 5000 ng/ml HMGB-1, respectively, or 25 ng/ml IL-8 for 2 h. Spontaneous migration was measured against 1% human serum in PBS (control) (for details, see Materials and Methods). Values represent means ± s.e.m. ( n = 5) and statistical difference between the control and each respective sample as determined using the Mann-Whitney test, * p < 0.05, ** p < 0.01.

Article Snippet: HMGB1 was initially identified using a polyclonal anti-HMGB1 from BD Biosciences Pharmingen (556528, BD Diagnostics, San Diego, CA) or anti-human HMGB1 (ab18256, Abcam, Cambridge, UK).

Techniques: Infection, Staining, Derivative Assay, Isolation, Enzyme-linked Immunosorbent Assay, Western Blot, Recombinant, Transwell Assay, Migration, MANN-WHITNEY

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: HMGB1 in severe soft tissue infections caused by Streptococcus pyogenes

doi: 10.3389/fcimb.2014.00004

Figure Lengend Snippet: Presence of pro-inflammatory mediators at the local site of tissue infection . The figure shows representative images of a snap-frozen cryo-sectioned biopsy, from a patient with severe streptococcal soft tissue infection. (A) Tissue sections were immunohistochemically stained for HMGB1, CXCL12, and IL-1β. A control staining, omitting the primary antibody was also made (control). Bars indicate 50 μm. Snap frozen tissue biopsies were also immunofluorescently stained for HMGB1 (green) in combination with (B) IL-1β (red) or (C) CXCL12 (red). Cell nuclei were stained blue with DAPI. Bars indicate 20 μm.

Article Snippet: HMGB1 was initially identified using a polyclonal anti-HMGB1 from BD Biosciences Pharmingen (556528, BD Diagnostics, San Diego, CA) or anti-human HMGB1 (ab18256, Abcam, Cambridge, UK).

Techniques: Infection, Staining

Journal: Cells

Article Title: Repeated Social Defeat Stress Induces HMGB1 Nuclear Export in Prefrontal Neurons, Leading to Social Avoidance in Mice.

doi: 10.3390/cells12131789

Figure Lengend Snippet: Figure 1. Intracerebroventricular infusion of recombinant HMGB1 suppresses repeated social defeat stress-induced social avoidance. (a) A scheme of the behavioral experiment. (b) The inhibitory effect of recombinant HMGB1 protein on the development of social avoidance by repeated social defeat stress. Mice in the Defeat group, which received the stress, and the Naïve group, which did not, were implanted with a cannula at the right lateral ventricle connected to an osmotic pump placed in the subcutaneous tissue of the back. The mice were kept isolated for 7 days before the stress (Day 1 to Day 10). Recombinant HMGB1 protein (5 ng/day) or saline (vehicle) had been infused throughout the experimental period from the cannula implantation. Data are shown as mean ± SEM. * p < 0.05, ** p < 0.01 between the mice infused with vehicle and recombinant HMGB1 protein in the Defeat group. $$$ p < 0.001 between the Naïve and Defeat groups with vehicle infusion.

Article Snippet: The primary antibodies used in this study were rat monoclonal anti-HMGB1 [8] (1:100 dilution), mouse monoclonal HMGB1 (MAB1690, R & D Systems, 1:100 dilution), mouse monoclonal αCaMKII (13-7300, Thermo Fisher Scientific, Waltham, MA, USA, 1:500 dilution), rabbit polyclonal GABA (A2052, Sigma-Aldrich, Saint Louis, MO, USA, 1:3000 dilution), rabbit polyclonal 5-HT (20080, Immunostar, 1:2000 dilution), and mouse monoclonal tyrosine hydroxylase (MAB318, Millipore, Darmstadt, Germany, 1:5000 dilution).

Techniques: Recombinant, Isolation, Saline

Journal: Cells

Article Title: Repeated Social Defeat Stress Induces HMGB1 Nuclear Export in Prefrontal Neurons, Leading to Social Avoidance in Mice.

doi: 10.3390/cells12131789

Figure Lengend Snippet: Figure 2. Intracerebroventricular infusion of neutralizing HMGB1 antibody promotes repeated social defeat stress-induced social avoidance. (a) A scheme of the behavioral experiment. (b) The facilitatory effect of neutralizing HMGB1 antibody on the development of social avoidance by repeated social defeat stress. Mice in the Defeat and Naïve groups were implanted with the cannula and osmotic pump, and neutralizing HMGB1 antibody (22.5 ng/day) or isotype control IgG was infused to the right lateral ventricles, as described in the legend of Figure 1. Data are shown as mean ± SEM. ** p < 0.01 between the mice infused with control IgG and neutralizing HMGB1 antibody in the Defeat group. $$$$ p < 0.0001 between the Naïve and Defeat groups with control IgG infusion. ### p < 0.001, #### p < 0.0001 between the Naïve and Defeat groups with neutralizing HMGB1 antibody infusion.

Article Snippet: The primary antibodies used in this study were rat monoclonal anti-HMGB1 [8] (1:100 dilution), mouse monoclonal HMGB1 (MAB1690, R & D Systems, 1:100 dilution), mouse monoclonal αCaMKII (13-7300, Thermo Fisher Scientific, Waltham, MA, USA, 1:500 dilution), rabbit polyclonal GABA (A2052, Sigma-Aldrich, Saint Louis, MO, USA, 1:3000 dilution), rabbit polyclonal 5-HT (20080, Immunostar, 1:2000 dilution), and mouse monoclonal tyrosine hydroxylase (MAB318, Millipore, Darmstadt, Germany, 1:5000 dilution).

Techniques: Control

Journal: Cells

Article Title: Repeated Social Defeat Stress Induces HMGB1 Nuclear Export in Prefrontal Neurons, Leading to Social Avoidance in Mice.

doi: 10.3390/cells12131789

Figure Lengend Snippet: Figure 3. mPFC infusion of the neutralizing HMGB1 antibody attenuates repeated social defeat stress- induced social avoidance. (a) A scheme of the behavioral experiment. (b) The inhibitory effect of neutralizing HMGB1 antibody on the development of social avoidance by repeated social defeat stress. Mice in the Naïve and Defeat groups were implanted with cannulas bilaterally at the mPFC connected to an osmotic pump placed in the subcutaneous tissue of the back. The mice were kept isolated for 7 days before the stress (Day 1 to Day 10). Neutralizing HMGB1 antibody (22.5 ng/day) or control IgG had been infused throughout the experimental period from the cannula implantation. Data are shown as mean ± SEM. * p < 0.05 between the Naïve and Defeat groups with control IgG infusion.

Article Snippet: The primary antibodies used in this study were rat monoclonal anti-HMGB1 [8] (1:100 dilution), mouse monoclonal HMGB1 (MAB1690, R & D Systems, 1:100 dilution), mouse monoclonal αCaMKII (13-7300, Thermo Fisher Scientific, Waltham, MA, USA, 1:500 dilution), rabbit polyclonal GABA (A2052, Sigma-Aldrich, Saint Louis, MO, USA, 1:3000 dilution), rabbit polyclonal 5-HT (20080, Immunostar, 1:2000 dilution), and mouse monoclonal tyrosine hydroxylase (MAB318, Millipore, Darmstadt, Germany, 1:5000 dilution).

Techniques: Isolation, Control

Journal: Cells

Article Title: Repeated Social Defeat Stress Induces HMGB1 Nuclear Export in Prefrontal Neurons, Leading to Social Avoidance in Mice.

doi: 10.3390/cells12131789

Figure Lengend Snippet: Figure 5. Repeated social defeat stress induces HMGB1 nuclear export in the mPFC of both susceptible and resilient mice. (a) A scheme of the behavioral experiment. The Naïve and Defeat mice were kept isolated for 7 days before repeated social defeat stress (Day 1 to Day 10). The mice were sacrificed at 90 min after an additional social defeat stress on Day 16 for HMGB1 immunostaining. Susceptible and resilient mice were chosen from the Defeat mice according to the behavioral criteria described in the Methods section. (b,c) Representative images of HMGB1 immunostaining in the mPFC of Naïve, susceptible, and resilient mice at low and high magnification, respectively. HMGB1 signals are shown in green. Nuclei were counterstained with Hoechst (red). Scale bar, 100 µm in (b) and 20 µm in (c). (d) The number of HMGB1-positive nuclei in the mPFC in the Naïve and Defeat groups. Individual dots represent the value of each mouse normalized to the average of the Naïve mice in each batch of experiments. Red and blue circles show the values of susceptible and resilient mice, respectively. Data are shown as mean ± SEM. * p < 0.05 for unpaired t-test. (e) Representative images of HMGB1 co-immunostaining with αCaMKII and GABA in the mPFC of Naïve and Defeat mice. HMGB1 signals are shown in green. αCaMKII and GABA are shown in red. Nuclei were counterstained with Hoechst (blue). Scale bar, 20 µm. (f) The percentage of HMGB1-positive nuclei in αCaMKII- or GABA-positive mPFC neurons of Naïve and Defeat mice. Data are shown as mean ± SEM. ** p < 0.01 between αCaMKII and GABA in the Naïve group.

Article Snippet: The primary antibodies used in this study were rat monoclonal anti-HMGB1 [8] (1:100 dilution), mouse monoclonal HMGB1 (MAB1690, R & D Systems, 1:100 dilution), mouse monoclonal αCaMKII (13-7300, Thermo Fisher Scientific, Waltham, MA, USA, 1:500 dilution), rabbit polyclonal GABA (A2052, Sigma-Aldrich, Saint Louis, MO, USA, 1:3000 dilution), rabbit polyclonal 5-HT (20080, Immunostar, 1:2000 dilution), and mouse monoclonal tyrosine hydroxylase (MAB318, Millipore, Darmstadt, Germany, 1:5000 dilution).

Techniques: Isolation, Immunostaining

Journal: Cells

Article Title: Repeated Social Defeat Stress Induces HMGB1 Nuclear Export in Prefrontal Neurons, Leading to Social Avoidance in Mice.

doi: 10.3390/cells12131789

Figure Lengend Snippet: Figure 6. RAGE deletion inhibits repeated social defeat stress-induced HMGB1 nuclear export. (a) A scheme of the behavioral experiment. Wild-type and RAGE-KO mice in the Naïve and Defeat group were subjected to behavioral experiments as described in the legend of Figure 5. (b) No effect of RAGE deletion on the development of social avoidance by repeated social defeat stress. (c,d) The inhibitory effect of RAGE deletion on HMGB1 nuclear export induced by repeated social defeat stress. Representative images (c) show HMGB1 immunofluorescent signals in the mPFC in wild-type and RAGE-KO mice of the Defeat group as well as wild-type mice of the Naïve group. HMGB1 signals are shown in green. Brain slices were counterstained with Neurotrace and Hoechst shown in blue and red, respectively. Scale bar, 20 µm. The number of HMGB1-positive nuclei of neurons in layers II/III of the mPFC (infralimbic (IL) and prelimbic (PrL) subregions, separately), normalized to the average of the Naïve mice in each batch of experiments, is shown (d). * p < 0.05 for Tukey’s multiple comparison for the indicated pairs.

Article Snippet: The primary antibodies used in this study were rat monoclonal anti-HMGB1 [8] (1:100 dilution), mouse monoclonal HMGB1 (MAB1690, R & D Systems, 1:100 dilution), mouse monoclonal αCaMKII (13-7300, Thermo Fisher Scientific, Waltham, MA, USA, 1:500 dilution), rabbit polyclonal GABA (A2052, Sigma-Aldrich, Saint Louis, MO, USA, 1:3000 dilution), rabbit polyclonal 5-HT (20080, Immunostar, 1:2000 dilution), and mouse monoclonal tyrosine hydroxylase (MAB318, Millipore, Darmstadt, Germany, 1:5000 dilution).

Techniques: Comparison

Journal: Acta pharmacologica Sinica

Article Title: Growth suppression and radiosensitivity increase by HMGB1 in breast cancer.

doi: 10.1111/j.1745-7254.2007.00669.x

Figure Lengend Snippet: Figure 1. HMGB1 binds to RB. (A) endogenous association of HMGB1 and RB. Nuclear extracts were prepared from MCF-7 and BT-549 cells and subjected to IP/Western blotting analyses. A mouse IgG IP from MCF-7 cells was used as the negative control. Antibodies used were: HMGB1 IP (a rabbit polyclonal antibody) and HMGB1 Western Blot (a mouse monoclonal antibody), RB IP (M-153), and RB Western Blot (C-15). Representative results are shown from 3 independent experiments. (B) In vitro interaction of HMGB1 and RB. GST capture assay was performed. 35S-methionine-labeled proteins were prepared by in vitro transcription and translation using the T3 promoter of mammalian expression pCMV-Tag2B vector that was used for cloning the full-length of wtHMGB1 or HMGB1-RXRXH cDNA. GST RB fusion proteins were generated from a RB cDNA cloned into a GST pGEX vector, expressed in Escheria coli, and purified by affinity chromatography (left panel). Input lanes show 10% IVT wtHMGB1 or 10% IVT HMGB1-RXRXH product used in the assay.

Article Snippet: Equal aliquots of protein extract (100 μg/lane) were electrophoresed on SDS-PAGE, transferred to nitrocellulose membranes (Millipore, Bedford, MA, USA), and blotted with primary antibodies: a mouse monoclonal anti-HMGB1 antibody (R&D Systems, Minneapolis, MN, USA); a rabbit polyclonal anti-RB antibody (C-15, Santa Cruz Biotechnology, USA) or a mixture of a rabbit polyclonal anti-PRAP antibody (P7607, Sigma, USA) plus a rabbit polyclonal anti-PRAP antibody (214/215, cleavage site specific antibody, Sigma, USA).

Techniques: Western Blot, Negative Control, In Vitro, Labeling, Expressing, Plasmid Preparation, Cloning, Generated, Clone Assay, Purification, Affinity Chromatography

Journal: Acta pharmacologica Sinica

Article Title: Growth suppression and radiosensitivity increase by HMGB1 in breast cancer.

doi: 10.1111/j.1745-7254.2007.00669.x

Figure Lengend Snippet: Figure 2. Enhancement of RB-mediated transcription repression by HMGB1. SAOS-2 cells were cotransfected with the indicated vectors (1 µg each vector/well in 6-well tissue culture dishes) by Lipofectamine 2000 for 24 h (A) or pre-incubated in 0.5% FCS medium for 24 h, cotransfected with the indicated vectors, and then incubated in 10% FCS medium for 24 h (B). Transfected cells were harvested for assay of luciferase activity. Total transfected DNA was kept constant by the addition of a pCMV-Tag2B control vector. A pRSV-β-gal plasmid was cotransfected as an internal standard for the normalization of luciferase values. Luciferase values are presented as mean±SEM from 3 independent experiments. Statistical significances were analyzed by two-tail Student’s t-test. cP<0.01.

Article Snippet: Equal aliquots of protein extract (100 μg/lane) were electrophoresed on SDS-PAGE, transferred to nitrocellulose membranes (Millipore, Bedford, MA, USA), and blotted with primary antibodies: a mouse monoclonal anti-HMGB1 antibody (R&D Systems, Minneapolis, MN, USA); a rabbit polyclonal anti-RB antibody (C-15, Santa Cruz Biotechnology, USA) or a mixture of a rabbit polyclonal anti-PRAP antibody (P7607, Sigma, USA) plus a rabbit polyclonal anti-PRAP antibody (214/215, cleavage site specific antibody, Sigma, USA).

Techniques: Plasmid Preparation, Incubation, Transfection, Luciferase, Activity Assay, Control

Journal: Acta pharmacologica Sinica

Article Title: Growth suppression and radiosensitivity increase by HMGB1 in breast cancer.

doi: 10.1111/j.1745-7254.2007.00669.x

Figure Lengend Snippet: Figure 3. HMGB1 causes RB-dependent suppression of cell proliferation. (A) time-course of recombinant adenovirus-directed expression of HMGB1. MCF-7 cells were infected with Ad5wtHMGB1 at a MOI of 100 pfu/cell. Protein extracts were prepared at the indicated times following infection; 50 µg protein from each lysate was resolved by SDS-PAGE and probed with a mouse monoclonal anti-HMGB1 antibody. As an internal control for the amount of protein loaded, the same filter was also immunoblotted with a polyclonal α-actin antibody (I-19). Immunoreactive proteins were visualized by ECL. (B) adenovirus-mediated expression of the HMGB1 gene inhibits MCF-7, but not BT-549 breast cancer cell proliferation. Cells were seeded at 3×104 cells/well in 6-well plates 1 d before infection with Ad5-wtHMGB1, Ad5-HMGB1- RXRXH, or Ad5-lacZ (a control) at a MOI of pfu/cell. At the indicated times, the cells in 10 wells were collected and counted by a hemocytometer. SEM from 10 wells in 3 independent experiments were less than 10%. Values for Ad5-wtHMGB1 were significantly different from those for Ad5-HMGB1–RXRXH and Ad-lacZ alone in MCF-7 (P<0.001, two-tail Student’s t-test).

Article Snippet: Equal aliquots of protein extract (100 μg/lane) were electrophoresed on SDS-PAGE, transferred to nitrocellulose membranes (Millipore, Bedford, MA, USA), and blotted with primary antibodies: a mouse monoclonal anti-HMGB1 antibody (R&D Systems, Minneapolis, MN, USA); a rabbit polyclonal anti-RB antibody (C-15, Santa Cruz Biotechnology, USA) or a mixture of a rabbit polyclonal anti-PRAP antibody (P7607, Sigma, USA) plus a rabbit polyclonal anti-PRAP antibody (214/215, cleavage site specific antibody, Sigma, USA).

Techniques: Recombinant, Expressing, Infection, SDS Page, Control

Journal: Acta pharmacologica Sinica

Article Title: Growth suppression and radiosensitivity increase by HMGB1 in breast cancer.

doi: 10.1111/j.1745-7254.2007.00669.x

Figure Lengend Snippet: Figure 4. HMGB1 inhibits cell growth in Rb+/+ MEF. Rb+/+ and Rb–/– MEF were infected with Ad5-wtHMGB1, Ad5-HMGB1- RXRXH, or Ad5-lacZ at a MOI of 100 pfu/cell for 92 h and counted by a hemocytometer. Error bars denote the SEM. HMGB1 protein levels determined by Western blot assay were shown to monitor HMGB1 expression after infection (left panel).

Article Snippet: Equal aliquots of protein extract (100 μg/lane) were electrophoresed on SDS-PAGE, transferred to nitrocellulose membranes (Millipore, Bedford, MA, USA), and blotted with primary antibodies: a mouse monoclonal anti-HMGB1 antibody (R&D Systems, Minneapolis, MN, USA); a rabbit polyclonal anti-RB antibody (C-15, Santa Cruz Biotechnology, USA) or a mixture of a rabbit polyclonal anti-PRAP antibody (P7607, Sigma, USA) plus a rabbit polyclonal anti-PRAP antibody (214/215, cleavage site specific antibody, Sigma, USA).

Techniques: Infection, Western Blot, Expressing

Journal: Acta pharmacologica Sinica

Article Title: Growth suppression and radiosensitivity increase by HMGB1 in breast cancer.

doi: 10.1111/j.1745-7254.2007.00669.x

Figure Lengend Snippet: Figure 5. Flow cytometry analysis of cell cycle and apoptosis. (A) distribution of MCF-7 cells in the cell cycle was analyzed by propidium iodide staining followed by flow cytometry after infection with Ad5-wtHMGB1, Ad5-HMGB1-RXRXH, or Ad5-lacZ (a control) at a MOI of 100 pfu/cell for 72 or 96 h. Representative histograms of 3 independent experiments are shown. Percentage of cells in sub-G1 is presented. (B) cells were uninfected or infected with Ad5-wtHMGB1, Ad5-HMGB1-RXRXH, or Ad5-lacZ (a control) for 92 h; cell extracts were then subjected to Western blotting using anti-PARP antibodies. (C) cells were infected with Ad5-wtHMGB1, Ad5-HMGB1-RXRXH or Ad5-lacZ (a control) for different incubation times. Caspase-3-like protease activity in cell lysates was assayed by spectrophometric detection of the chromophore p-nitroanilide after cleavage from the labeled substrate DEVD-p-nitroanilide. bP<0.05, cP<0.01 compared with control values. Mean±SEM from 3 independent experiments.

Article Snippet: Equal aliquots of protein extract (100 μg/lane) were electrophoresed on SDS-PAGE, transferred to nitrocellulose membranes (Millipore, Bedford, MA, USA), and blotted with primary antibodies: a mouse monoclonal anti-HMGB1 antibody (R&D Systems, Minneapolis, MN, USA); a rabbit polyclonal anti-RB antibody (C-15, Santa Cruz Biotechnology, USA) or a mixture of a rabbit polyclonal anti-PRAP antibody (P7607, Sigma, USA) plus a rabbit polyclonal anti-PRAP antibody (214/215, cleavage site specific antibody, Sigma, USA).

Techniques: Flow Cytometry, Staining, Infection, Control, Western Blot, Incubation, Activity Assay, Labeling

Journal: Acta pharmacologica Sinica

Article Title: Growth suppression and radiosensitivity increase by HMGB1 in breast cancer.

doi: 10.1111/j.1745-7254.2007.00669.x

Figure Lengend Snippet: Figure 7. HMGB1 suppresses tumorigenicity in athymic nude mice. Exponentially growing MCF-7 and BT-549 cells were infected in vitro at a MOI of 100 pfu/cell with Ad5-wtHMGB1, Ad5-HMGB1- RXRXH, Ad5-HMGB1∆LXCXE, or Ad5-LacZ (as a control) 48 h before subcutaneous injections into the left flank of 5 week old fe- male nu/nu mice (10 mice each injection). Tumor diameters were monitored weekly. Mean tumor volumes±SEM from 2 independent experiments are shown. ANOVA was performed to determine statis- tical significance between each treatment group using Statistica software; P<0.05 was considered significant.

Article Snippet: Equal aliquots of protein extract (100 μg/lane) were electrophoresed on SDS-PAGE, transferred to nitrocellulose membranes (Millipore, Bedford, MA, USA), and blotted with primary antibodies: a mouse monoclonal anti-HMGB1 antibody (R&D Systems, Minneapolis, MN, USA); a rabbit polyclonal anti-RB antibody (C-15, Santa Cruz Biotechnology, USA) or a mixture of a rabbit polyclonal anti-PRAP antibody (P7607, Sigma, USA) plus a rabbit polyclonal anti-PRAP antibody (214/215, cleavage site specific antibody, Sigma, USA).

Techniques: Infection, In Vitro, Control, Injection, Software

Journal: Inflammation Research

Article Title: Crocin alleviates lipopolysaccharide-induced acute respiratory distress syndrome by protecting against glycocalyx damage and suppressing inflammatory signaling pathways

doi: 10.1007/s00011-019-01314-z

Figure Lengend Snippet: Effects of crocin on HMGB1 and NF-κB pathway activation in LPS-induced ARDS mice and LPS-stimulated HUVECs. Following the process shown in Fig. a, the levels of HMGB1 and NF-κB p65 as well as the phosphorylation and non-phosphorylation of IκBα in lung tissues ( a ) and HUVECs ( b ) were detected by Western blot. c , d Protein quantification of a , b , respectively. GAPDH or Lamin B1 was used as internal control. All data are presented as means ± SD of three independent experiments. # p < 0.05 vs. control group, * p < 0.05 vs. LPS group

Article Snippet: Rabbit monoclonal antibody to HMGB1 (ab79823), NF-κB p65 (ab32536), IκBα (ab32518), p-IκBα (ab133462), JNK (ab112501), ERK (ab184699), p38 (ab170099), p-ERK (ab201015), p-JNK (ab4821), p-p38 (ab47363), LaminB1 (ab133741), MMP-9 (ab38898), HPA (ab85543), SDC-4 (ab24511), Ly6g (ab25377), β-actin (ab8224), GAPDH (ab181602), MMP-9 inhibitor (MMP-9 inhib, ab142180), and FITC (ab25539) were acquired from Abcam Trading Company Ltd.

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Control

Journal: Frontiers in Immunology

Article Title: CBP Bromodomain Inhibition Rescues Mice From Lethal Sepsis Through Blocking HMGB1-Mediated Inflammatory Responses

doi: 10.3389/fimmu.2020.625542

Figure Lengend Snippet: SGC-CBP30 treatment prevented LPS-induced lethal endotoxemia and CLP-induced sepsis. (A) Male BALB/c mice received SGC-CBP30 (19.3 mg/kg; intraperitoneal injection) 30 min or 8 h after a lethal dose of LPS (10 mg/kg; intraperitoneal injection). Similar treatments with DEX (1.3 mg/kg; intraperitoneal injection) or saline were taken as control. Mouse survival rate was monitored continuously for 72 h after LPS treatment. Blood was collected after 18 h, and serum HMGB1 (B) and TNF-α (C) were determined by ELISA. (D) Male BALB/c mice were subjected to CLP and SGC-CBP30 (19.3 mg/kg; intraperitoneal injection) was administrated to the mice 30 min or 8 h after surgical procedure. Survival of mice was monitored for 72 h. Serum levels of HMGB1 (E) and TNF-α (F) 18 h after CLP were measured using ELISA. In (A, D) , Kaplan-Meier analysis was used to analyze the survival rate of septic mice. n=10 mice/group. In (B, C, E, F) , graphs show mean ± SD. n=6 mice/group. *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: Recombinant human HMGB1 (rhHMGB1, 1690-HMB) and antibody against HMGB1 (MAB16901) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Injection, Saline, Control, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: CBP Bromodomain Inhibition Rescues Mice From Lethal Sepsis Through Blocking HMGB1-Mediated Inflammatory Responses

doi: 10.3389/fimmu.2020.625542

Figure Lengend Snippet: SGC-CBP30 plus ciprofloxacin combination therapy protected mice against lethal sepsis. (A) Male BALB/c mice were administered ciprofloxacin (7.5 mg/kg) intravenously at 1 h after CLP, and thereafter administered SGC-CBP30 (19.3 mg/kg) at 8 h after CLP. Survival of mice was monitored for up to 72 h. At 18 h post the onset of sepsis, serum levels of HMGB1 (B) and TNF-α (C) were determined by ELISA. (D) Mice were euthanized 18 h after surgeries and selected organs were collected. Lung, colon, liver, kidney of septic mice was stained with hematoxylin and eosin, and examined by light microscopy (×400). Scale bar: 100 μm. Histological scoring of tissue injury was evaluated as described in the Materials and Methods section. For lung tissues, the following parameters were evaluated: (a) alveolar septal thickness, (b) interstitial edema, (c) infiltration of inflammatory cells, (d) alveolar congestion/collapse; colon tissues: (a) bleeding ulcers in the intestinal mucosa, (b) interstitial edema, (c) infiltration of inflammatory cells, (d) disorganized architecture with intestinal gland; liver tissues: (a) centrilobular necrosis, (b) hepatocyte edema, (c) infiltration of inflammatory cells, (d) central venous congestion; kidney tissues: (a) epithelial cell brush-border loss, (b) interstitial edema, (c) infiltration of inflammatory cells, (d) glomerular shrinkage. In (a), Kaplan-Meier analysis was used to analyze the survival rates of septic mice. n=10 mice/group. In (B–D) , graphs show mean ± SD. n=5 or 6 mice/group. ***p < 0.001.

Article Snippet: Recombinant human HMGB1 (rhHMGB1, 1690-HMB) and antibody against HMGB1 (MAB16901) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Staining, Light Microscopy

Journal: Frontiers in Immunology

Article Title: CBP Bromodomain Inhibition Rescues Mice From Lethal Sepsis Through Blocking HMGB1-Mediated Inflammatory Responses

doi: 10.3389/fimmu.2020.625542

Figure Lengend Snippet: Inhibitory effect of SGC-CBP30 on HMGB1 release and expression in LPS-stimulated cells. THP-1 cells (A) and primary MPM cells (B) were treated with SGC-CBP30 (4 μM) at 8 h after stimulation with LPS (500 ng/ml). After LPS stimulation for the indicated time, HMGB1 release was measured by ELISA. THP-1 cells were treated with LPS (500 ng/ml) for 8 h and then incubated with SGC-CBP30 (4 μM). After the indicated time of LPS stimulation, nuclear and cytoplasmic fractions were analyzed by Western blot using anti-HMGB1 antibody (C) . Cells were incubated with mouse anti-HMGB1 antibody and then incubated with Alexa Flour 555-conjugated anti-mouse (red) secondary antibody. The nuclei were counterstained with DAPI (blue). The location of HMGB1 was observed under a confocal laser microscope. Scale bar: 10 μm (D) . The cell lysates were immunoprecipitated with anti-HMGB1 antibody, followed by immunoblotting with anti-acetyl lysine and anti-HMGB1 antibodies (E) . Quantification of HMGB1 transcripts by real-time PCR with GAPDH as the internal control (F) . Whole cell lysates were subjected to immunoblotting with anti-HMGB1 and anti-β-actin antibodies (G) . Data shown were representative of three independent experiments. Error bars indicate mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: Recombinant human HMGB1 (rhHMGB1, 1690-HMB) and antibody against HMGB1 (MAB16901) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Incubation, Western Blot, Microscopy, Immunoprecipitation, Real-time Polymerase Chain Reaction, Control

Journal: Frontiers in Immunology

Article Title: CBP Bromodomain Inhibition Rescues Mice From Lethal Sepsis Through Blocking HMGB1-Mediated Inflammatory Responses

doi: 10.3389/fimmu.2020.625542

Figure Lengend Snippet: Effects of CBP suppression on HMGB1-mediated inflammatory response. THP-1 cells (A) and primary MPM cells (B) were pretreated with SGC-CBP30 (4 μM) for 2 h and then stimulated with rhHMGB1 (500 ng/ml) for 12 h; the production of TNF-α, IL-1β, and IL-6 were measured by ELISA. (C) THP-1 cells and primary MPM cells were pretreated with or without SGC-CBP30 (4 μM) for 2 h followed by stimulation with rhHMGB1 (100 ng/ml) for 1 h. Cell lysates were prepared and the levels of phospho-MAPKs, phospho-IKKα/β and phospho-IκBα were determined by Western blot analysis. (D) THP-1 cells were transfected with CBP shRNA or negative control shRNA, and after 72 h, stimulated with rhHMGB1 (100 ng/ml) for 1 h. The cell lysates were subjected to immunoblotting analysis using indicated antibodies. Data shown were representative of three independent experiments. Error bars indicate mean ± SD. **p < 0.01; ***p < 0.001.

Article Snippet: Recombinant human HMGB1 (rhHMGB1, 1690-HMB) and antibody against HMGB1 (MAB16901) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Transfection, shRNA, Negative Control

Journal: Frontiers in Immunology

Article Title: CBP Bromodomain Inhibition Rescues Mice From Lethal Sepsis Through Blocking HMGB1-Mediated Inflammatory Responses

doi: 10.3389/fimmu.2020.625542

Figure Lengend Snippet: Schematic illustration of CBP bromodomain inhibitor prevents sepsis-related HMGB1 secretion and blocks HMGB1 pro-inflammatory activity. The potent and selective CBP inhibitor, SGC-CBP30, significantly rescues mice from LPS-/CLP-induced sepsis model. In addition, SGC-CBP30 suppresses LPS-induced HMGB1 expression and cytoplasmic translocation. Furthermore, by triggering the reactivation of PP2A and the stabilization of MKP-1, CBP bromodomain inhibition prevents rhHMGB1-stimulated activation of MAPKs and NF-κB pathways and production of proinflammatory cytokines.

Article Snippet: Recombinant human HMGB1 (rhHMGB1, 1690-HMB) and antibody against HMGB1 (MAB16901) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Activity Assay, Expressing, Translocation Assay, Inhibition, Activation Assay