Journal: Journal of Inflammation (London, England)

Article Title: Fas (CD95) induces rapid, TLR4/IRAK4-dependent release of pro-inflammatory HMGB1 from macrophages

doi: 10.1186/1476-9255-7-30

Figure Lengend Snippet: Fas activation induces rapid release of HMGB1 from primary murine peritoneal mϕ and RAW264.7 cells . (A) HMGB1 in cell culture-conditioned medium was detected by immunoblot as described in Materials and Methods following treatment with recombinant mFasL (0 - 0.5 μg/ml) for 2 hr. (B) HMGB1 in cell culture-conditioned medium was detected by immunoblot as described in Materials and Methods following treatment with mFasL, mAb Jo2 and LPS respectively (0.25 μg/ml for each stimulus) for the times indicated (0 - 24 hr). (C) HMGB1 in cell culture-conditioned media from primary wild-type and Fas lpr murine peritoneal mϕs culture-conditioned medium was detected by immunoblot as described in Materials and Methods following treatment with mFasL, mAb Jo2 and LPS (0.25 μg/ml for each stimulus), respectively, for the times indicated (0 - 24 hr). All the results are representative of 3 independent experiments that yielded similar results.

Article Snippet: After blocking with 5% non-fat milk at room temperature for 1 hr, the transfer membranes were incubated for 1 hr with primary antibodies specific for HMGB1 (Abcam, MA, USA), proliferating cell nuclear antigen (PCNA; BD Biosciences, CA, USA) and β-actin (Cell Signaling, Pickering, ON, CANADA), respectively.

Techniques: Activation Assay, Cell Culture, Western Blot, Recombinant

Journal: Journal of Inflammation (London, England)

Article Title: Fas (CD95) induces rapid, TLR4/IRAK4-dependent release of pro-inflammatory HMGB1 from macrophages

doi: 10.1186/1476-9255-7-30

Figure Lengend Snippet: Fas activation induces cytoplamsic translocation of HMGB1 in primary murine peritoneal mϕ . Primary murine peritoneal mϕ were stimulated with recombinant mFasL (0 - 0.5 μg/ml) for 2 hr. Cytoplasmic and nuclear extracts were prepared as described in Materials and Methods, and HMGB1 content was determined by immunoblot. Equal loading of samples was confirmed by concomitant immunoblot analysis of the respective fractions using antibodies specific for a nuclear (PCNA) or cytoplasmic (β-actin) protein. Blots are representative of 3 independent experiments with similar results.

Article Snippet: After blocking with 5% non-fat milk at room temperature for 1 hr, the transfer membranes were incubated for 1 hr with primary antibodies specific for HMGB1 (Abcam, MA, USA), proliferating cell nuclear antigen (PCNA; BD Biosciences, CA, USA) and β-actin (Cell Signaling, Pickering, ON, CANADA), respectively.

Techniques: Activation Assay, Translocation Assay, Recombinant, Western Blot

Journal: Journal of Inflammation (London, England)

Article Title: Fas (CD95) induces rapid, TLR4/IRAK4-dependent release of pro-inflammatory HMGB1 from macrophages

doi: 10.1186/1476-9255-7-30

Figure Lengend Snippet: Neutralization of HMGB1 decreases Fas-induced pro-inflammatory cytokine production by mϕ . Primary murine peritoneal mϕ were pretreated with anti-HMGB1 mAb (0.25 μg/ml) or isotype control IgG2b (0.25 μg/ml) for 1 hr, prior to stimulation with mFasL (0.25 μg/ml) for the times indicated (0 - 18 hr) as described in Materials and Methods. Pro-inflammatory cytokine production was determined by measurement of (A) TNF-α (4A) and (B) MIP-2 (4B) in culture supernatants by cytokine-specific ELISAs as described in Materials and Methods. Data are presented as mean ± SD of 3 independent experiments, n = 3/group. * P ≤ 0.05; ** P ≤ 0.01.

Article Snippet: After blocking with 5% non-fat milk at room temperature for 1 hr, the transfer membranes were incubated for 1 hr with primary antibodies specific for HMGB1 (Abcam, MA, USA), proliferating cell nuclear antigen (PCNA; BD Biosciences, CA, USA) and β-actin (Cell Signaling, Pickering, ON, CANADA), respectively.

Techniques: Neutralization, Control

Journal: Journal of Inflammation (London, England)

Article Title: Fas (CD95) induces rapid, TLR4/IRAK4-dependent release of pro-inflammatory HMGB1 from macrophages

doi: 10.1186/1476-9255-7-30

Figure Lengend Snippet: Fas-induced release of HMGB1 by primary murine peritoneal mϕ is TLR4/IRAK4-dependent . Primary murine peritoneal mϕ derived from (A) wild-type vs Tlr2 -/- vs Tlr4 -/- mice, or (B) wild-type vs Irak4 -/- mice, were stimulated with mFasL (0.25 μg/ml) for the times indicated (0 - 6 hr) as described in Materials and Methods. HMGB1 release into the concentrated cell culture-conditioned medium was detected by immunoblot as described in Materials and Methods. Results are representative of 3 independent experiments that yielded similar results.

Article Snippet: After blocking with 5% non-fat milk at room temperature for 1 hr, the transfer membranes were incubated for 1 hr with primary antibodies specific for HMGB1 (Abcam, MA, USA), proliferating cell nuclear antigen (PCNA; BD Biosciences, CA, USA) and β-actin (Cell Signaling, Pickering, ON, CANADA), respectively.

Techniques: Derivative Assay, Cell Culture, Western Blot

Journal: Journal of cellular and molecular medicine

Article Title: HDAC4/5-HMGB1 signalling mediated by NADPH oxidase activity contributes to cerebral ischaemia/reperfusion injury.

doi: 10.1111/jcmm.12040

Figure Lengend Snippet: Fig. 2 Expression patterns of HDACs in ischaemic brain of rats subjected to focal cerebral ischaemia reperfusion. (A) Real-time RT-PCR analysis of HDAC1-11 mRNA levels from total RNAs extracted from ischaemic core and penumbra in the brain, among Zn2+-dependent HDACs (HDAC1-11), mRNA levels of HDAC4 and HDAC5 were significantly decreased; HDAC9 expression was remarkably increased. (B) Representative Western blot gel documents and summarized data showing the protein levels of HDAC1-11 in the ischaemic core and penumbra of the ischaemic brain. (C) Immuno- histochemistry for HDAC4 and HDAC5 at 24 hrs of reperfusion after MCAO in ischaemic core and penumbra in the brain. (D) Representative Wes- tern blot gel documents and summarized data showing the protein levels of HDAC4 and HDAC5 in the ischaemic core and penumbra of the brain at different reperfusion time-points. (E) Immunofluorescent staining showing cellular localization of HDAC4 in the cortex from ischaemic brain and nor- mal subjects, indicating that HDAC4 was expressed in neurons rather than astrocytes and microglia. *P < 0.05 versus sham-operated rats (n = 8).

Article Snippet: Primary antibodies to – HDAC1, HDAC2, HDAC8, HDAC9, HDAC11, HMGB1 and NOX2 (1:1000 dilution; abcam, Cambridge, MA, USA), HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, and HDAC10 (1:1000 dilution, ProteinTech Group, Chicago, IL, USA), and secondary antibodies horseradish peroxidase-labelled antimouse IgG or antirabbit IgG (1:6000 dilution, ProteinTech Group) were used in this study.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunohistochemistry, Staining

Journal: Journal of cellular and molecular medicine

Article Title: HDAC4/5-HMGB1 signalling mediated by NADPH oxidase activity contributes to cerebral ischaemia/reperfusion injury.

doi: 10.1111/jcmm.12040

Figure Lengend Snippet: Fig. 3 Regulation of HDAC4/HDAC5 and HMGB1 expressions is associated with NADPH oxidase activity in PC12 cells by OGD. (A) Representative Western blot gel documents and summarized data showing NOX2 protein levels in PC12 cells cultured by the model of OGD. (B) Summarized data showing the effect of apocynin on NADPH oxidase activity in PC12 cells. (C) Representative Western blot gel docu- ments and summarized data showing the expression levels of HDAC4, HDAC5 and HMGB1 in PC12 cells. *P < 0.05 versus control, #P < 0.05 versus cells cultured by the model of OGD (n = 6).

Article Snippet: Primary antibodies to – HDAC1, HDAC2, HDAC8, HDAC9, HDAC11, HMGB1 and NOX2 (1:1000 dilution; abcam, Cambridge, MA, USA), HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, and HDAC10 (1:1000 dilution, ProteinTech Group, Chicago, IL, USA), and secondary antibodies horseradish peroxidase-labelled antimouse IgG or antirabbit IgG (1:6000 dilution, ProteinTech Group) were used in this study.

Techniques: Activity Assay, Western Blot, Cell Culture, Expressing, Control

Journal: Journal of cellular and molecular medicine

Article Title: HDAC4/5-HMGB1 signalling mediated by NADPH oxidase activity contributes to cerebral ischaemia/reperfusion injury.

doi: 10.1111/jcmm.12040

Figure Lengend Snippet: Fig. 4 The effects of NADPH oxidase-mediated HDAC4/5 signalling on the expression and release of HMGB1. (A) Quantitative RT-PCR analysis of HDAC4 mRNA level in pCMV6-HDAC4 transfected PC12 cells. (B) Representative Western blot gel documents and summarized data showing the rel- ative HDAC4 expression in pCMV6-HDAC4 transfected PC12 cells. (C) Quantitative RT-PCR analysis of HDAC5 mRNA level in pCMV6-HDAC5 trans- fected PC12 cells. (D) Representative Western blot gel documents and summarized data showing the relative HDAC5 expression in pCMV6-HDAC5 transfected PC12 cells. (E) Representative Western blot gel documents and summarized data showing the effect of NADPH oxidase inhibitor apocy- nin, HDAC4 and HDAC5 on the expression of HMGB1 in PC12 cells cultured by the model of OGD. (F) Summarized data showing the release of HMGB1 in the cell culture supernatant measured by ELISA. (G) Summarized data showing cell apoptosis determined by flow cytometric analysis indicating that HDAC4 and HDAC5 protect cell from death in PC12 cells by OGD. *P < 0.05 versus control, #P < 0.05 versus cells cultured by the model of OGD, &P < 0.05 versus cells cultured by the model of OGD and apocynin treatment (n = 6).

Article Snippet: Primary antibodies to – HDAC1, HDAC2, HDAC8, HDAC9, HDAC11, HMGB1 and NOX2 (1:1000 dilution; abcam, Cambridge, MA, USA), HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, and HDAC10 (1:1000 dilution, ProteinTech Group, Chicago, IL, USA), and secondary antibodies horseradish peroxidase-labelled antimouse IgG or antirabbit IgG (1:6000 dilution, ProteinTech Group) were used in this study.

Techniques: Expressing, Quantitative RT-PCR, Transfection, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay, Control

Journal: Journal of cellular and molecular medicine

Article Title: HDAC4/5-HMGB1 signalling mediated by NADPH oxidase activity contributes to cerebral ischaemia/reperfusion injury.

doi: 10.1111/jcmm.12040

Figure Lengend Snippet: Fig. 5 Inhibition of NADPH oxidase activity ameliorates cerebral ischaemia/reperfusion injury and decreased HDAC4/HDAC5 and HMGB1 release. (A) Representative photo- graphs of TTC staining and calculated infarct volume in rats with apocynin treat- ment after cerebral ischaemia/reperfusion. (B) Neurological deficit scores in rats with apocynin treatment after cerebral ischae- mia/reperfusion. (C) Summarized data showing the effect of apocynin on NADPH oxidase activity in core and penumbra of the brain. (D) Representative Western blot gel documents and summarized data showing the protein levels of HDAC4 and HDAC5 in core and penumbra of the brain. (E) The HMGB1 levels in cerebro- spinal fluid were determined using ELISA. (F) Serum levels of HMGB1 at 24 hrs after reperfusion were determined using ELISA. *P < 0.05 versus sham-operated rats, #P < 0.05 versus ischaemic rats (n = 8).

Article Snippet: Primary antibodies to – HDAC1, HDAC2, HDAC8, HDAC9, HDAC11, HMGB1 and NOX2 (1:1000 dilution; abcam, Cambridge, MA, USA), HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, and HDAC10 (1:1000 dilution, ProteinTech Group, Chicago, IL, USA), and secondary antibodies horseradish peroxidase-labelled antimouse IgG or antirabbit IgG (1:6000 dilution, ProteinTech Group) were used in this study.

Techniques: Inhibition, Activity Assay, Staining, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: Neutrophils recruited to immunization sites initiating vaccine-induced antibody responses by locally expressing BAFF

doi: 10.1016/j.isci.2022.104453

Figure Lengend Snippet: Relationship between neutrophils recruited to rCPdA-E immunization sites and the local macrophages and B cells Mice (n = 3 in each group) were i.p. immunized once on day 0, followed PLCs were collected at 0.5 and 6 h postimmunization and analyzed for the expression of chemokines and B cell activation factors and B cell activation by qRT-PCR or flow cytometry. Peritoneal macrophages and bone marrow neutrophils were isolated from naive mice and stimulated with rCPdA or rCPdA-E for 6 h followed by analyzing their expression of CXCL2 and BAFF mRNA by qRT-PCR. (A) The mRNA expression of neutrophil chemokines and B cell activation factors in PLCs. (B) CXCL2 and BAFF producing cells in PLCs. (C) The percentages of BAFF + Ly6G + , BAFF + CD19 + cells and the expression of CD69 and CD40 on CD19 + B cells in PLCs. (D) The mRNA expression of Cxcl2 and Baff in cultured macrophages. (E) The mRNA expression of Cxcl2 and Baff in cultured neutrophils. The data were the representative mean (mean ± SD) values of at least three independent experiments. Data were analyzed by unpaired t -test and ANOVAs. ∗, p < 0.05 ，∗∗, p < 0.01 ，∗∗∗, p < 0.001 , ∗∗∗∗, p < 0.0001 .

Article Snippet: For direct and surface staining, the isolated cells were stained with the fluorescence conjugated monoclonal antibodies against mouse Ly6G (clone 1A8; BD), CD19 (clone 1D3; BD), CD69 (clone H1.2F3; BD), CD40 (clone 3/23; BD), F4/80 (clone T45-2342; BD), which were purchased from BD Biosciences, at 4°C in dark for 30 min. For indirect intracellular staining and detecting His-tagged antigen uptake, the cells were first fixed with 4% paraformaldehyde and permeabilized with 0.1% saponin for 10 min, then blocked with the normal serum (5% v/v) from the host species for 30 min, followed by staining with anti-HMGB1 (ab18256, Abcam), anti-CXCL2 (orb10749, Biorbyt) and anti-His-tag (ab9108, Abcam) monoclonal antibodies for 30 min. After being washed twice with FACS buffer (PBS containing 0.5% BSA and 2 mM EDTA), incubated the cells with Alexa Fluor 488-conjugated goat-anti-rabbit (ab150077, Abcam) or APC-conjugated goat-anti-rabbit (ab130805, Abcam) secondary antibody for 30 min. For direct intracellular molecule staining, the fixed and permeabilized cells were stained with anti-TLR9 (clone 1138D; R&D), anti-IRF5 (clone W16007B; Biolegend) and anti-BAFF (orb495712, Biorbyt) antibodies at 4°C in dark for 30 min.

Techniques: Expressing, Activation Assay, Quantitative RT-PCR, Flow Cytometry, Isolation, Cell Culture

Journal: iScience

Article Title: Neutrophils recruited to immunization sites initiating vaccine-induced antibody responses by locally expressing BAFF

doi: 10.1016/j.isci.2022.104453

Figure Lengend Snippet: Expression of TLR9 and IRF5 in PLCs and spleens of immunized mice Mice (n = 3 in each group) were i.p. immunized with rCPdA or rCPdA-E once and then their PLCs and spleens were collected for detecting the expression of TLR9-IRF5 signaling molecules either by qRT-PCR at mRNA level or western blotting and flow cytometry at protein level. (A) The mRNA levels of TLR9-IRF5 signaling molecules in PLCs of the mice at 0.5 and 6h post-immunization. (B) The protein levels of TLR9 and IRF5 in PLCs of the mice at 6 h postimmunization. The expression of TLR9. (C) and IRF5. (D) in Ly6G + and CD19 + PLCs of the mice at 6h post-immunization. The expression of TLR9. (E) and IIRF5. (F) in CD19 + splenocytes (spls) of the mice at 6h postimmunization. Student’s unpaired t -test was used to determine the statistical significance of two groups and ANOVAs were used to determine the multiple comparisons. Data represent mean ± SD All the experiments were repeated three times. p< 0.05 ， ∗∗, p < 0.01 ， ∗∗∗, p < 0.001, ∗∗∗∗, p < 0.0001 .

Article Snippet: For direct and surface staining, the isolated cells were stained with the fluorescence conjugated monoclonal antibodies against mouse Ly6G (clone 1A8; BD), CD19 (clone 1D3; BD), CD69 (clone H1.2F3; BD), CD40 (clone 3/23; BD), F4/80 (clone T45-2342; BD), which were purchased from BD Biosciences, at 4°C in dark for 30 min. For indirect intracellular staining and detecting His-tagged antigen uptake, the cells were first fixed with 4% paraformaldehyde and permeabilized with 0.1% saponin for 10 min, then blocked with the normal serum (5% v/v) from the host species for 30 min, followed by staining with anti-HMGB1 (ab18256, Abcam), anti-CXCL2 (orb10749, Biorbyt) and anti-His-tag (ab9108, Abcam) monoclonal antibodies for 30 min. After being washed twice with FACS buffer (PBS containing 0.5% BSA and 2 mM EDTA), incubated the cells with Alexa Fluor 488-conjugated goat-anti-rabbit (ab150077, Abcam) or APC-conjugated goat-anti-rabbit (ab130805, Abcam) secondary antibody for 30 min. For direct intracellular molecule staining, the fixed and permeabilized cells were stained with anti-TLR9 (clone 1138D; R&D), anti-IRF5 (clone W16007B; Biolegend) and anti-BAFF (orb495712, Biorbyt) antibodies at 4°C in dark for 30 min.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Flow Cytometry

Journal: iScience

Article Title: Neutrophils recruited to immunization sites initiating vaccine-induced antibody responses by locally expressing BAFF

doi: 10.1016/j.isci.2022.104453

Figure Lengend Snippet:

Article Snippet: For direct and surface staining, the isolated cells were stained with the fluorescence conjugated monoclonal antibodies against mouse Ly6G (clone 1A8; BD), CD19 (clone 1D3; BD), CD69 (clone H1.2F3; BD), CD40 (clone 3/23; BD), F4/80 (clone T45-2342; BD), which were purchased from BD Biosciences, at 4°C in dark for 30 min. For indirect intracellular staining and detecting His-tagged antigen uptake, the cells were first fixed with 4% paraformaldehyde and permeabilized with 0.1% saponin for 10 min, then blocked with the normal serum (5% v/v) from the host species for 30 min, followed by staining with anti-HMGB1 (ab18256, Abcam), anti-CXCL2 (orb10749, Biorbyt) and anti-His-tag (ab9108, Abcam) monoclonal antibodies for 30 min. After being washed twice with FACS buffer (PBS containing 0.5% BSA and 2 mM EDTA), incubated the cells with Alexa Fluor 488-conjugated goat-anti-rabbit (ab150077, Abcam) or APC-conjugated goat-anti-rabbit (ab130805, Abcam) secondary antibody for 30 min. For direct intracellular molecule staining, the fixed and permeabilized cells were stained with anti-TLR9 (clone 1138D; R&D), anti-IRF5 (clone W16007B; Biolegend) and anti-BAFF (orb495712, Biorbyt) antibodies at 4°C in dark for 30 min.

Techniques: Recombinant, Software

Journal: iScience

Article Title: Neutrophils recruited to immunization sites initiating vaccine-induced antibody responses by locally expressing BAFF

doi: 10.1016/j.isci.2022.104453

Figure Lengend Snippet:

Article Snippet: For direct and surface staining, the isolated cells were stained with the fluorescence conjugated monoclonal antibodies against mouse Ly6G (clone 1A8; BD), CD19 (clone 1D3; BD), CD69 (clone H1.2F3; BD), CD40 (clone 3/23; BD), F4/80 (clone T45-2342; BD), which were purchased from BD Biosciences, at 4°C in dark for 30 min. For indirect intracellular staining and detecting His-tagged antigen uptake, the cells were first fixed with 4% paraformaldehyde and permeabilized with 0.1% saponin for 10 min, then blocked with the normal serum (5% v/v) from the host species for 30 min, followed by staining with anti-HMGB1 (ab18256, Abcam), anti-CXCL2 (orb10749, Biorbyt) and anti-His-tag (ab9108, Abcam) monoclonal antibodies for 30 min. After being washed twice with FACS buffer (PBS containing 0.5% BSA and 2 mM EDTA), incubated the cells with Alexa Fluor 488-conjugated goat-anti-rabbit (ab150077, Abcam) or APC-conjugated goat-anti-rabbit (ab130805, Abcam) secondary antibody for 30 min. For direct intracellular molecule staining, the fixed and permeabilized cells were stained with anti-TLR9 (clone 1138D; R&D), anti-IRF5 (clone W16007B; Biolegend) and anti-BAFF (orb495712, Biorbyt) antibodies at 4°C in dark for 30 min.

Techniques: Recombinant, Software