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  • 99
    Millipore hmgb1
    Trauma/hemorrhagic shock induces ER stress in the intestinal epithelium in a TLR4-dependent manner, causing enterocyte apoptosis and the release of <t>HMGB1</t>
    Hmgb1, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 522 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    hmgb1  (Abcam)
    92
    Abcam hmgb1
    RAGE blockage reduces CDT-mediated inflammatory responses. (A) AGS cells were pretreated with RAGE antagonist (2 μM RAP) for 2 h before incubation with 100 nM CDT for 24 h. Cell lysates were analyzed by western blotting with the antibodies against RAGE, <t>HMGB1,</t> and β-actin, respectively. The protein expression of RAGE (B) and HMGB1 (C) was quantified by densitometric analysis and normalized to β-actin. (D) Cells were co-transfected with NF- κ B - and pGL3-luciferase reporters prior to treatment with the 2 μM RAP followed by exposure to 100 nM CDT for 24 h. pGL3-luciferase reporter was used for monitoring transfection efficiency. NF- κ B promoter activity was determined and normalized by pGL3 luciferase activity. (E) The cell culture supernatant was prepared to evaluate IL-8 production using ELISA. The data are presented as means ± standard deviations for three independent experiments. Statistical analysis was calculated using ANOVA analysis and Tukey's test. * P
    Hmgb1, supplied by Abcam, used in various techniques. Bioz Stars score: 92/100, based on 3298 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems hmgb1
    The <t>HMGB1</t> expression increased in fetal membranes after IA LPS exposure: photomicrographs (40× objective) of the immunohistochemistry for HMGB1 showed no baseline staining of the chorioamnion in the saline control (A) with increased expression amnion (AM) and chorion (CH) at 24 hours after intra-amniotic LPS (B), with further increases throughout the time course to 15 days after IA LPS (C-E). Data for (F) and (H) are shown as box (25th-75th percentile) and whisker (10th-90th percentile) plot with horizontal line in each box depicting the median. The mRNA quantitation of HMGB1 by reverse transcription-polymerase chain reaction (RT-PCR) showing no change in expression after IA LPS (F). Western blot of HMGB1 showed increased expression at 24 hours to 2 days after intra-amniotic LPS (G-H; * P
    Hmgb1, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 1177 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Shino-Test Corporation hmgb1
    Regulation of <t>HMGB1‐TLR4</t> signalling pathway on DCsHMGB1 activates DCs. Comparison of expression of CD80 and CD86 in mDCs among all groups. After isolation, culture, purification and identification of DCs, the expression of mDCs costimulatory molecules was detected by flow cytometry. A, The geometric mean optical density of CD80 in DCs; (B) the geometric mean optical density of CD86 in DCs. Comparison of 24, 48, 72 h concentrations of IL‐12, TNF‐α, IL‐8, IL‐6, in the supernatant among all groups. C, the changes of concentration of IL‐12 in the supernatant at 24, 48 and 72 h in each group; (D) the changes of concentration of TNF‐α in the supernatant at 24, 48 and 72 h in each group; (E) the changes of concentration of IL‐8 in the supernatant at 24, 48 and 72 h in each group; (F) the changes of concentration of IL‐6 in the supernatant at 24, 48 and 72 h in each group. Comparison of 24, 48, and 72 h protein and mRNA levels of MyD88, NF‐κB of mDCs from different groups. G, 24, 48, and 72 h Western blot analysis of MyD88 protein expression; (H) 24, 48, and 72 h Western blot analysis of NF‐κB protein expression; (I) 24, 48, and 72 h relative expression of MyD88 mRNA; (J) 24, 48, and 72 h relative expression of NF‐κB mRNA. * P
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    Cell Signaling Technology Inc hmgb1
    <t>HMGB1</t> aggravated macrophage inflammatory response. (a-b) RAW264.7 cells were stimulated with ALD-DNA (0, 25, 50 μ g/mL) for 24 h, levels of HMGB1 in the supernatants of RAW264.7 cells were analyzed by ELISA (a) and western blot analysis (b). Data are means ± SD of three independent experiments. (c-d) The efficiency of HMGB1 overexpression (c) and knockdown (d) was monitored by representative immunoblot of three independent experiments in ALD-DNA-stimulated RAW264.7 cells. (e-f) RAW264.7 cells were transfected with pHMGB1 or vector. 72 h after transfection, RAW264.7 cells were stimulated with PBS, UnALD-DNA or ALD-DNA (50 μ g/mL) followed by analyzing the levels of TNF- α (e) and IL-6 (f) in the culture supernatants of RAW264.7 cells. Data are means ± SD of three independent experiments. (g-h) RAW264.7 cells were transfected with control siRNA (200 nM) or HMGB1 siRNA (siHMGB1, 200 nM). After 72 h RAW264.7 cells were stimulated with PBS, UnALD-DNA or ALD-DNA (50 μ g/mL). ELISA assay was used to analyze the levels of TNF- α (g) and IL-6 (h) in the culture supernatants of RAW264.7 cells. Data are means ± SD of three independent experiments. (i-j) CD11b + /F4/80 high renal macrophages were sorted from nephritic single-cell suspensions from (i) pHMGB1- or empty vector-treated, (j) glycyrrhizin- or PBS-treated SLE mice by flow cytometry. Macrophages (2 × 10 5 /mL) were stimulated with ALD-DNA (50 μ g/mL) for 24 h. The supernatants were collected and assayed for the TNF- α and IL-6 concentrations using ELISA. Data are means ± SD from 8 mice in each group. ∗ P
    Hmgb1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 2183 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology hmgb1
    The relative expression of <t>HMGB1</t> and HMGN2 mRNA in gingival tissues from the patients in CP, G-AgP and healthy groups. A. Values were presented as mean ± SD for at least three independent experiments performed in triplicate. The expressions of HMGB1 and HMGN2 in control group were normalized in 1.0. B. Representative RT-PCR images for HMGB1 and HMGN2 in four groups.
    Hmgb1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 391 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti hmgb1
    Suppression of systemic inflammatory aggravation in PSI animals by blocking <t>HMGB1</t> function. a Animals were administered HMGB1 A box (5 μg/kg) or HPep1 (5 μg/kg) intranasally 3 h prior to LPS administration (100 μg/kg, i.p.) at 24 h post-MCAO. b , c Serum HMGB1 levels were examined at 2 or 4 days post-MCAO for MCAO or PSI animals with or without A box or HPep1 treatment at 21 h post-MCAO by immunoblotting. d , e Levels of TNF-α and IL-1β in the serum were measured in MCAO, MCAO+LPS, MCAO+LPS+A box (5 μg/kg), or MCAO+LPS+HPep1 (5 μg/kg) group at 2 days post-MCAO by ELISA. Results are presented as means ± SEMs ( n = 4). Sham or S sham-operated rats, LPS LPS-treated rats, MCAO treatment-naive MCAO rats, MCAO+LPS LPS-treated MCAO rats, MCAO+LPS+A box A box-treated MCAO+LPS rats, MCAO+LPS+HPep1 HPep1-treated MCAO+LPS rats. ** p
    Anti Hmgb1, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 763 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    HMGBiotech hmgb1
    Effect of <t>HMGB1</t> and IL-1β on the levels of MMP released into the medium by osteoarthritic synoviocytes . (a) Matrix metalloproteinase (MMP)-1, (b) MMP-3 and (c) MMP-13 protein levels in the medium. Cells were stimulated with IL-1b (10 ng/ml) for 24 hours in the presence or absence of high mobility group box 1 (HMGB1) at 15 and 25 ng/ml. MMP protein was measured by ELISA in supernatants. Data are expressed as mean ± standard error of the mean. Duplicate samples from six to eight patients were used. ++ P
    Hmgb1, supplied by HMGBiotech, used in various techniques. Bioz Stars score: 93/100, based on 291 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam anti hmgb1 antibody
    Effects of siRNA on 8-nitroG formation in indium-treated A549 cells. ( A ) Reduction in <t>HMGB1,</t> RAGE and TLR9 expression by siRNA transfection into A549 cells. Effects of siRNA on protein expression were evaluated by Western blotting. These blots were cropped from different parts in the same gel, and each blot was divided with white lines. Full-length blots are shown in Supplementary Figure S2 online. ( B ) Image analysis for HMGB1, RAGE and TLR9 expression in siRNA-transfected A549 cells. These values were expressed as fold changes compared with control. ( C ) Fluorescent images of 8-nitroG formation in indium-treated A549 cells and effects of siRNA. Cells were transfected with 10 nM siRNA for HMGB1 , AGER and TLR9 or negative control siRNA for 2 days and then treated with 200 ng/ml indium compounds for 4 h as described in “ Methods ” section. 8-NitroG formation was evaluated by immunocytochemistry as described in “ Methods ” section. The nucleus was stained with Hoechst 33258. Magnification, × 200. ( D ) Quantitative image analysis for the effects of siRNA on 8-nitroG formation in indium-treated A549 cells. Staining intensities of 8-nitroG per area were analyzed with an image J software. The relative intensity of the control was set at 1. ( B , D ) The data were expressed as means ± SD of 3–4 independent experiments. ** p
    Anti Hmgb1 Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 515 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher hmgb1
    Conditional knockout of <t>HMGB1</t> in rods does not affect microglial activation or mobilization. ( A ) Retinas of HMGB1ΔRod and control mice were sectioned at a 30-µm thickness and probed with anti–Iba-1 antibody to view activation and mobilization of microglia/macrophage at 7 dprd. Nuclei were counterstained with DAPI. Scale bar : 50 µm. ( B ) Entire retinas of HMGB1ΔRod and control mice were probed with anti–Iba-1 antibody and flat-mounted for confocal imaging at 7 dprd. Multiple confocal images were Z-stacked to create merged images of different layers of the retina based on the DAPI staining. Scale bar : 200 µm. ( C ) Supernatants from eyecups were collected and the levels of HMGB1 in HMGB1ΔRod and control mice at 3 and 7 dprd were measured by ELISA. One-way ANOVA ( n = 4). IPL, inner plexiform layer; IS/OS, inner and outer segment; ns, not significant; OPL, outer plexiform layer.
    Hmgb1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 182 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Epitomics hmgb1
    (a) The expression of <t>HMGB1,</t> TNF- α , and IL-6 on cDNA level was detected by real-time PCR ( * P
    Hmgb1, supplied by Epitomics, used in various techniques. Bioz Stars score: 92/100, based on 125 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam rabbit anti hmgb1
    Blood <t>HMGB1</t> levels increase before epilepsy onset in electrical SE–exposed rats and predict therapeutic response to antiinflammatory drugs. Longitudinal analysis of total HMGB1 and levels of acetylated, reduced, and disulfide isoforms in blood plasma at representative time points of disease development in SE-exposed rats receiving treatment (drug) or corresponding vehicle (see key). Treatment included anakinra+BoxA+ifenprodil (SE+drug) (detailed protocol in Methods; Supplemental Figure 5A ). Data are shown as mean ± SEM ( n = 9 rats each group; dot plots are shown in Supplemental Figure 7 ). Rats are the same as reported in Figure 3 . * P
    Rabbit Anti Hmgb1, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 451 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Chondrex inc hmgb1 protein
    Interference of <t>HMGB1</t> expression with siRNA
    Hmgb1 Protein, supplied by Chondrex inc, used in various techniques. Bioz Stars score: 89/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GenePharma Company hmgb1
    <t>HMGB1</t> knockdown-induced apoptosis of RCC cells. Notes: ( A ) Apoptosis in A498 cells transfected with HMGB1-shRNA, Con-shRNA, and the indicated vectors were analyzed by flow cytometry. ( B ) Apoptosis in ACHN cells transfected with HMGB1-siRNA, Con-siRNA, and the indicated vectors were analyzed by flow cytometry. The results are representative of 3 separate experiments. ** P
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    Shino-Test Corporation hmgb1 elisa kit
    Effect of JNK inhibition on TNF-α or <t>TNF-α/HMGB1-induced</t> myocyte apoptosis. Cardiomyocytes were pretreated with the JNK inhibitor (SP-600125; 10 μM) for 30 min and subsequently exposed to M199 or M199 containing either TNF-α (40 ng/ml) or TNF-α/HMGB1 (1 μg/ml) cocktail. After a 24-h incubation period, cardiomyocyte apoptosis was assessed with caspase-3 activity ( top ) and a cell death <t>ELISA</t> kit ( bottom ). Values are means ± SE; n = 3. * P
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    GeneTex hmgb1
    Survival curves of high-mobility group box 1 and vascular endothelial growth factor C in intrahepatic cholangiocarcinoma. Kaplan-Meier method univariate analyses of <t>HMGB1</t> (A); VEGF-C (B); Survival curves of intrahepatic cholangiocarcinoma patients with
    Hmgb1, supplied by GeneTex, used in various techniques. Bioz Stars score: 92/100, based on 66 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam antibody against hmgb1
    SAHA induced down-regulation of NF- κ B1 was <t>HMGB1</t> dependent. LX2 cells were transfected by siR-NC or siR-HMGB1 48 h before 2.5 µM SAHA treatment. (A) The mRNA expression of NF- κ B1 (p50) in each group was detected by RT-PCR. (B) Protein expressions of p50 and p65 in each group were determined by western blot assay. (C) Dual luciferase reporter assay. The activity of NF- κ B in each group was detected by a dual luciferase reporter system. NF- κ B driven firefly luciferase activity was assayed by a dual luciferase reporter system, renilla luciferase activity served as internal control, the results were expressed as relative luciferase activity. The mRNA or protein expressions were normalized to GAPDH. ∗∗ P
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    Abcam anti hmgb1 antibody epr3507
    SAHA induced down-regulation of NF- κ B1 was <t>HMGB1</t> dependent. LX2 cells were transfected by siR-NC or siR-HMGB1 48 h before 2.5 µM SAHA treatment. (A) The mRNA expression of NF- κ B1 (p50) in each group was detected by RT-PCR. (B) Protein expressions of p50 and p65 in each group were determined by western blot assay. (C) Dual luciferase reporter assay. The activity of NF- κ B in each group was detected by a dual luciferase reporter system. NF- κ B driven firefly luciferase activity was assayed by a dual luciferase reporter system, renilla luciferase activity served as internal control, the results were expressed as relative luciferase activity. The mRNA or protein expressions were normalized to GAPDH. ∗∗ P
    Anti Hmgb1 Antibody Epr3507, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 89 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Assay Designs Inc hmgb1
    Cardiac cells express the <t>HMGB1</t> receptors: RAGE and TLR4 receptor-4. These expressions were evaluated at the mRNA level [reverse transcriptase–polymerase chain reaction ( A )] and at the protein level [western immunoblotting ( B )]
    Hmgb1, supplied by Assay Designs Inc, used in various techniques. Bioz Stars score: 92/100, based on 54 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Trauma/hemorrhagic shock induces ER stress in the intestinal epithelium in a TLR4-dependent manner, causing enterocyte apoptosis and the release of HMGB1

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Intestinal epithelial Toll-like Receptor-4 activation is required for the development of acute lung injury after trauma/hemorrhagic shock via the release of HMGB1 from the gut

    doi: 10.4049/jimmunol.1402490

    Figure Lengend Snippet: Trauma/hemorrhagic shock induces ER stress in the intestinal epithelium in a TLR4-dependent manner, causing enterocyte apoptosis and the release of HMGB1

    Article Snippet: To further support a role for HMGB1 in the development of lung injury, injection of recombinant HMGB1 (4μg/g body weight) into TLR4ΔIEC mice (which themselves had showed reduced HMGB1 levels in the plasma ) reversed the protection from trauma induced lung injury that had been seen in the TLR4ΔIEC mice, which under these conditions displayed significant lung inflammation after trauma ( ; the corresponding serum levels of HMGB1 are shown in ).

    Techniques:

    Remote lung injury after trauma requires HMGB1 release from the intestinal epithelium

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Intestinal epithelial Toll-like Receptor-4 activation is required for the development of acute lung injury after trauma/hemorrhagic shock via the release of HMGB1 from the gut

    doi: 10.4049/jimmunol.1402490

    Figure Lengend Snippet: Remote lung injury after trauma requires HMGB1 release from the intestinal epithelium

    Article Snippet: To further support a role for HMGB1 in the development of lung injury, injection of recombinant HMGB1 (4μg/g body weight) into TLR4ΔIEC mice (which themselves had showed reduced HMGB1 levels in the plasma ) reversed the protection from trauma induced lung injury that had been seen in the TLR4ΔIEC mice, which under these conditions displayed significant lung inflammation after trauma ( ; the corresponding serum levels of HMGB1 are shown in ).

    Techniques:

    HMGB1 signaling in the intestinal epithelium leads to internalization of the tight junction protein ZO-1 in the pulmonary epithelium

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Intestinal epithelial Toll-like Receptor-4 activation is required for the development of acute lung injury after trauma/hemorrhagic shock via the release of HMGB1 from the gut

    doi: 10.4049/jimmunol.1402490

    Figure Lengend Snippet: HMGB1 signaling in the intestinal epithelium leads to internalization of the tight junction protein ZO-1 in the pulmonary epithelium

    Article Snippet: To further support a role for HMGB1 in the development of lung injury, injection of recombinant HMGB1 (4μg/g body weight) into TLR4ΔIEC mice (which themselves had showed reduced HMGB1 levels in the plasma ) reversed the protection from trauma induced lung injury that had been seen in the TLR4ΔIEC mice, which under these conditions displayed significant lung inflammation after trauma ( ; the corresponding serum levels of HMGB1 are shown in ).

    Techniques:

    Trauma/hemorrhagic shock induces TLR4 signaling in the intestine leading to lung injury via the release of HMGB1 from the intestine

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Intestinal epithelial Toll-like Receptor-4 activation is required for the development of acute lung injury after trauma/hemorrhagic shock via the release of HMGB1 from the gut

    doi: 10.4049/jimmunol.1402490

    Figure Lengend Snippet: Trauma/hemorrhagic shock induces TLR4 signaling in the intestine leading to lung injury via the release of HMGB1 from the intestine

    Article Snippet: To further support a role for HMGB1 in the development of lung injury, injection of recombinant HMGB1 (4μg/g body weight) into TLR4ΔIEC mice (which themselves had showed reduced HMGB1 levels in the plasma ) reversed the protection from trauma induced lung injury that had been seen in the TLR4ΔIEC mice, which under these conditions displayed significant lung inflammation after trauma ( ; the corresponding serum levels of HMGB1 are shown in ).

    Techniques:

    RAGE blockage reduces CDT-mediated inflammatory responses. (A) AGS cells were pretreated with RAGE antagonist (2 μM RAP) for 2 h before incubation with 100 nM CDT for 24 h. Cell lysates were analyzed by western blotting with the antibodies against RAGE, HMGB1, and β-actin, respectively. The protein expression of RAGE (B) and HMGB1 (C) was quantified by densitometric analysis and normalized to β-actin. (D) Cells were co-transfected with NF- κ B - and pGL3-luciferase reporters prior to treatment with the 2 μM RAP followed by exposure to 100 nM CDT for 24 h. pGL3-luciferase reporter was used for monitoring transfection efficiency. NF- κ B promoter activity was determined and normalized by pGL3 luciferase activity. (E) The cell culture supernatant was prepared to evaluate IL-8 production using ELISA. The data are presented as means ± standard deviations for three independent experiments. Statistical analysis was calculated using ANOVA analysis and Tukey's test. * P

    Journal: Frontiers in Immunology

    Article Title: Coalescence of RAGE in Lipid Rafts in Response to Cytolethal Distending Toxin-Induced Inflammation

    doi: 10.3389/fimmu.2019.00109

    Figure Lengend Snippet: RAGE blockage reduces CDT-mediated inflammatory responses. (A) AGS cells were pretreated with RAGE antagonist (2 μM RAP) for 2 h before incubation with 100 nM CDT for 24 h. Cell lysates were analyzed by western blotting with the antibodies against RAGE, HMGB1, and β-actin, respectively. The protein expression of RAGE (B) and HMGB1 (C) was quantified by densitometric analysis and normalized to β-actin. (D) Cells were co-transfected with NF- κ B - and pGL3-luciferase reporters prior to treatment with the 2 μM RAP followed by exposure to 100 nM CDT for 24 h. pGL3-luciferase reporter was used for monitoring transfection efficiency. NF- κ B promoter activity was determined and normalized by pGL3 luciferase activity. (E) The cell culture supernatant was prepared to evaluate IL-8 production using ELISA. The data are presented as means ± standard deviations for three independent experiments. Statistical analysis was calculated using ANOVA analysis and Tukey's test. * P

    Article Snippet: Danger-associated molecular pattern (DAMP) proteins, such as HMGB1, S100, IL-1α, and IL-33/ST2, are endogenous danger signals ( – ).

    Techniques: Incubation, Western Blot, Expressing, Transfection, Luciferase, Activity Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

    CDT induces RAGE expression and proinflammatory cytokine production in the mouse small intestine. (A) Mice were treated with PBS or CDT (2.5 mg/kg) by intragastric gavage once every 2 days for six administrations. Arrows in red indicated the days of CDT administration. (B) Tissue sections of the jejunum were prepared and fixed in 4% paraformaldehyde and subjected to hematoxylin-eosin (H E) or immunohistochemical (IHC) staining with antibodies against RAGE, HMGB1, IL-1β, TNF-α, and IL-6, respectively. The magnified images are shown in the right panel of each cropped area. Arrows in yellow represented severe infiltration of inflammatory cells in the intestinal epithelium with pathological derangement. Pronounced expression of proinflammatory cytokines shown in intestinal tissues were indicated by red arrows. Scale bars in left panels, 20 μm and in magnified right panels, 200 μm.

    Journal: Frontiers in Immunology

    Article Title: Coalescence of RAGE in Lipid Rafts in Response to Cytolethal Distending Toxin-Induced Inflammation

    doi: 10.3389/fimmu.2019.00109

    Figure Lengend Snippet: CDT induces RAGE expression and proinflammatory cytokine production in the mouse small intestine. (A) Mice were treated with PBS or CDT (2.5 mg/kg) by intragastric gavage once every 2 days for six administrations. Arrows in red indicated the days of CDT administration. (B) Tissue sections of the jejunum were prepared and fixed in 4% paraformaldehyde and subjected to hematoxylin-eosin (H E) or immunohistochemical (IHC) staining with antibodies against RAGE, HMGB1, IL-1β, TNF-α, and IL-6, respectively. The magnified images are shown in the right panel of each cropped area. Arrows in yellow represented severe infiltration of inflammatory cells in the intestinal epithelium with pathological derangement. Pronounced expression of proinflammatory cytokines shown in intestinal tissues were indicated by red arrows. Scale bars in left panels, 20 μm and in magnified right panels, 200 μm.

    Article Snippet: Danger-associated molecular pattern (DAMP) proteins, such as HMGB1, S100, IL-1α, and IL-33/ST2, are endogenous danger signals ( – ).

    Techniques: Expressing, Mouse Assay, Immunohistochemistry, Staining

    CDT induces RAGE and HMGB1 expression. (A) AGS cells were exposed to CDT for 24 h at various concentrations (0–500 nM), and (B) treated with 100 nM CDT at different time points (0–48 h). Total cell lysates were prepared to measure the expression of RAGE and HMGB1 by western blotting, and β-actin was used as the protein loading control. Protein expression levels of RAGE and HMGB1 were quantified by densitometric analysis and normalized to β-actin, respectively (B–F) . The data are presented as means ± standard deviations for three independent experiments. Statistical analysis was calculated using ANOVA analysis and Tukey's test. * P

    Journal: Frontiers in Immunology

    Article Title: Coalescence of RAGE in Lipid Rafts in Response to Cytolethal Distending Toxin-Induced Inflammation

    doi: 10.3389/fimmu.2019.00109

    Figure Lengend Snippet: CDT induces RAGE and HMGB1 expression. (A) AGS cells were exposed to CDT for 24 h at various concentrations (0–500 nM), and (B) treated with 100 nM CDT at different time points (0–48 h). Total cell lysates were prepared to measure the expression of RAGE and HMGB1 by western blotting, and β-actin was used as the protein loading control. Protein expression levels of RAGE and HMGB1 were quantified by densitometric analysis and normalized to β-actin, respectively (B–F) . The data are presented as means ± standard deviations for three independent experiments. Statistical analysis was calculated using ANOVA analysis and Tukey's test. * P

    Article Snippet: Danger-associated molecular pattern (DAMP) proteins, such as HMGB1, S100, IL-1α, and IL-33/ST2, are endogenous danger signals ( – ).

    Techniques: Expressing, Western Blot

    Disruption of lipid rafts decreases CDT-induced inflammatory response. (A) AGS cells were pretreated with or without 10 μM lovastatin for 1 h and exposed to 100 nM CDT for 24 h. Cell lysates were analyzed by western blotting with the antibodies against RAGE, HMGB1, and β-actin, respectively. AGS cells were co-transfected with NF- κ B and pGL3 luciferase reporters in the absence or presence of 10 μM lovastatin before treatment of 100 nM CDT for 24 h. Cell lysates were used to analyze (B) NF- κ B promoter activity and normalized by pGL3 luciferase activity. (C) Cell supernatants were subjected to ELISA for the quantification of IL-8 production. The data are presented as means ± standard deviations for three independent experiments. Statistical analysis was calculated using ANOVA analysis and Tukey's test. * P

    Journal: Frontiers in Immunology

    Article Title: Coalescence of RAGE in Lipid Rafts in Response to Cytolethal Distending Toxin-Induced Inflammation

    doi: 10.3389/fimmu.2019.00109

    Figure Lengend Snippet: Disruption of lipid rafts decreases CDT-induced inflammatory response. (A) AGS cells were pretreated with or without 10 μM lovastatin for 1 h and exposed to 100 nM CDT for 24 h. Cell lysates were analyzed by western blotting with the antibodies against RAGE, HMGB1, and β-actin, respectively. AGS cells were co-transfected with NF- κ B and pGL3 luciferase reporters in the absence or presence of 10 μM lovastatin before treatment of 100 nM CDT for 24 h. Cell lysates were used to analyze (B) NF- κ B promoter activity and normalized by pGL3 luciferase activity. (C) Cell supernatants were subjected to ELISA for the quantification of IL-8 production. The data are presented as means ± standard deviations for three independent experiments. Statistical analysis was calculated using ANOVA analysis and Tukey's test. * P

    Article Snippet: Danger-associated molecular pattern (DAMP) proteins, such as HMGB1, S100, IL-1α, and IL-33/ST2, are endogenous danger signals ( – ).

    Techniques: Western Blot, Transfection, Luciferase, Activity Assay, Enzyme-linked Immunosorbent Assay

    RAGE binding of S100P, S100A4, and HMGB-1 was inhibited by an S100P-derived RAGE antagonistic peptide (RAP)

    Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

    Article Title: S100P-DERIVED RAGE ANTAGONISTIC PEPTIDE (RAP) REDUCES TUMOR GROWTH AND METASTASIS

    doi: 10.1158/1078-0432.CCR-12-0221

    Figure Lengend Snippet: RAGE binding of S100P, S100A4, and HMGB-1 was inhibited by an S100P-derived RAGE antagonistic peptide (RAP)

    Article Snippet: RAGE and HMGB-1 were expressed in both normal and cancer cells.

    Techniques: Binding Assay, Derivative Assay

    Pancreatic cancer cells expressed and were stimulated by RAGE and its ligands S100P, S100A4, and HMGB-1

    Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

    Article Title: S100P-DERIVED RAGE ANTAGONISTIC PEPTIDE (RAP) REDUCES TUMOR GROWTH AND METASTASIS

    doi: 10.1158/1078-0432.CCR-12-0221

    Figure Lengend Snippet: Pancreatic cancer cells expressed and were stimulated by RAGE and its ligands S100P, S100A4, and HMGB-1

    Article Snippet: RAGE and HMGB-1 were expressed in both normal and cancer cells.

    Techniques:

    The HMGB1 expression increased in fetal membranes after IA LPS exposure: photomicrographs (40× objective) of the immunohistochemistry for HMGB1 showed no baseline staining of the chorioamnion in the saline control (A) with increased expression amnion (AM) and chorion (CH) at 24 hours after intra-amniotic LPS (B), with further increases throughout the time course to 15 days after IA LPS (C-E). Data for (F) and (H) are shown as box (25th-75th percentile) and whisker (10th-90th percentile) plot with horizontal line in each box depicting the median. The mRNA quantitation of HMGB1 by reverse transcription-polymerase chain reaction (RT-PCR) showing no change in expression after IA LPS (F). Western blot of HMGB1 showed increased expression at 24 hours to 2 days after intra-amniotic LPS (G-H; * P

    Journal: Reproductive Sciences

    Article Title: Damage-Associated Molecular Pattern and Fetal Membrane Vascular Injury and Collagen Disorganization in Lipopolysaccharide-Induced Intra-amniotic Inflammation in Fetal Sheep

    doi: 10.1177/1933719115594014

    Figure Lengend Snippet: The HMGB1 expression increased in fetal membranes after IA LPS exposure: photomicrographs (40× objective) of the immunohistochemistry for HMGB1 showed no baseline staining of the chorioamnion in the saline control (A) with increased expression amnion (AM) and chorion (CH) at 24 hours after intra-amniotic LPS (B), with further increases throughout the time course to 15 days after IA LPS (C-E). Data for (F) and (H) are shown as box (25th-75th percentile) and whisker (10th-90th percentile) plot with horizontal line in each box depicting the median. The mRNA quantitation of HMGB1 by reverse transcription-polymerase chain reaction (RT-PCR) showing no change in expression after IA LPS (F). Western blot of HMGB1 showed increased expression at 24 hours to 2 days after intra-amniotic LPS (G-H; * P

    Article Snippet: Membranes were blocked with 5% Blotto (Santa Cruz Biotechnology, Inc, Dallas, TX, USA) in Tris-buffered saline with Tween and then incubated overnight with HMGB1 (MAB1690; R & D systems) at 1:1000 or mouse monoclonal anti-α SMA (A5228; Sigma) at 1:2500 or antismooth muscle myosin heavy chain 11 (ab53219) at 1:1000 or anti β-actin (ab6276; Abcam) at 1:10 000 or anti-α tubulin (ab18251; Abcam) at 1:5000 antibody dilutions.

    Techniques: Expressing, IA, Immunohistochemistry, Staining, Whisker Assay, Quantitation Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot

    Regulation of HMGB1‐TLR4 signalling pathway on DCsHMGB1 activates DCs. Comparison of expression of CD80 and CD86 in mDCs among all groups. After isolation, culture, purification and identification of DCs, the expression of mDCs costimulatory molecules was detected by flow cytometry. A, The geometric mean optical density of CD80 in DCs; (B) the geometric mean optical density of CD86 in DCs. Comparison of 24, 48, 72 h concentrations of IL‐12, TNF‐α, IL‐8, IL‐6, in the supernatant among all groups. C, the changes of concentration of IL‐12 in the supernatant at 24, 48 and 72 h in each group; (D) the changes of concentration of TNF‐α in the supernatant at 24, 48 and 72 h in each group; (E) the changes of concentration of IL‐8 in the supernatant at 24, 48 and 72 h in each group; (F) the changes of concentration of IL‐6 in the supernatant at 24, 48 and 72 h in each group. Comparison of 24, 48, and 72 h protein and mRNA levels of MyD88, NF‐κB of mDCs from different groups. G, 24, 48, and 72 h Western blot analysis of MyD88 protein expression; (H) 24, 48, and 72 h Western blot analysis of NF‐κB protein expression; (I) 24, 48, and 72 h relative expression of MyD88 mRNA; (J) 24, 48, and 72 h relative expression of NF‐κB mRNA. * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: The role of dendritic cells regulated by HMGB1/TLR4 signalling pathway in myocardial ischaemia reperfusion injury, et al. The role of dendritic cells regulated by HMGB1/TLR4 signalling pathway in myocardial ischaemia reperfusion injury

    doi: 10.1111/jcmm.14192

    Figure Lengend Snippet: Regulation of HMGB1‐TLR4 signalling pathway on DCsHMGB1 activates DCs. Comparison of expression of CD80 and CD86 in mDCs among all groups. After isolation, culture, purification and identification of DCs, the expression of mDCs costimulatory molecules was detected by flow cytometry. A, The geometric mean optical density of CD80 in DCs; (B) the geometric mean optical density of CD86 in DCs. Comparison of 24, 48, 72 h concentrations of IL‐12, TNF‐α, IL‐8, IL‐6, in the supernatant among all groups. C, the changes of concentration of IL‐12 in the supernatant at 24, 48 and 72 h in each group; (D) the changes of concentration of TNF‐α in the supernatant at 24, 48 and 72 h in each group; (E) the changes of concentration of IL‐8 in the supernatant at 24, 48 and 72 h in each group; (F) the changes of concentration of IL‐6 in the supernatant at 24, 48 and 72 h in each group. Comparison of 24, 48, and 72 h protein and mRNA levels of MyD88, NF‐κB of mDCs from different groups. G, 24, 48, and 72 h Western blot analysis of MyD88 protein expression; (H) 24, 48, and 72 h Western blot analysis of NF‐κB protein expression; (I) 24, 48, and 72 h relative expression of MyD88 mRNA; (J) 24, 48, and 72 h relative expression of NF‐κB mRNA. * P

    Article Snippet: 2.16 Experimental grouping and processing of the in vitro experiments mDCs was randomly divided into five groups, each group had three time points of 24, 48 and 72 hours, with three wells in each group. (a) C group: control group, mDCs was added to the complete medium of RPMI l640; (b) H group: HMGB1 (Ab215008; Abcam) was added to the complete medium containing mDCs (Ab215008, Abcam) (1 μg/mL); (c) H‐H‐Ig group: HMGB1 (1ug/mL) was added followed by HMGB1 specific neutralizing antibody (ST326052233; Shino‐Test, Tokyo, Japan) (the final concentration was 1 μg/mL) was added 30 minutes later; (d) H‐TLR4‐A group: HMGB1(1ug/ml) was added followed by TLR4 antagonists (A3850; ApexBio, Boston, MA) (the final concentration was 1 μg/mL) 30 minutes later; (e) H‐Ig group: HMGB1 (1ug/mL) was added followed by control IgG antibody (ST326058471; Shino‐Test) (the final concentration was 1 μg/mL) 30 minutes later.

    Techniques: Expressing, Isolation, Purification, Flow Cytometry, Cytometry, Concentration Assay, Western Blot

    HMGB1‐TLR4 signalling pathway mediates the migration, adhesion and activation of dendritic cells (DCs) in ischaemia‐reperfusion myocardium. A, Double immunofluorescence of DCs in myocardial tissue among all groups (n = 10 for each group). Samples were collected right after IR procedure and staining was performed as described in Section 2 . Representative fluorescence images (200×) of the distribution of CD1a (green), of CD80 (red). Arrows indicate co‐localization. (B,C) The geometric mean optical density of CD80, CD86 among all groups (n = 10 for each group) in peripheral blood of rats was detected by flow cytometry. Histological study, immunohistochemical staining in cardiac tissues from rats of different groups (n = 10 for each group). Samples were collected right after IR procedure was over and staining. D, Hematoxylin‐eosin (HE) staining pictures (200×) are shown in the left, immunohistochemical staining pictures (200×) are in the middle and right; (E) integral optical density (IOD) of ICAM‐1; (F) IOD of E‐selectin; (G) IOD of P‐selectin. Scale bars, 100 μm. * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: The role of dendritic cells regulated by HMGB1/TLR4 signalling pathway in myocardial ischaemia reperfusion injury, et al. The role of dendritic cells regulated by HMGB1/TLR4 signalling pathway in myocardial ischaemia reperfusion injury

    doi: 10.1111/jcmm.14192

    Figure Lengend Snippet: HMGB1‐TLR4 signalling pathway mediates the migration, adhesion and activation of dendritic cells (DCs) in ischaemia‐reperfusion myocardium. A, Double immunofluorescence of DCs in myocardial tissue among all groups (n = 10 for each group). Samples were collected right after IR procedure and staining was performed as described in Section 2 . Representative fluorescence images (200×) of the distribution of CD1a (green), of CD80 (red). Arrows indicate co‐localization. (B,C) The geometric mean optical density of CD80, CD86 among all groups (n = 10 for each group) in peripheral blood of rats was detected by flow cytometry. Histological study, immunohistochemical staining in cardiac tissues from rats of different groups (n = 10 for each group). Samples were collected right after IR procedure was over and staining. D, Hematoxylin‐eosin (HE) staining pictures (200×) are shown in the left, immunohistochemical staining pictures (200×) are in the middle and right; (E) integral optical density (IOD) of ICAM‐1; (F) IOD of E‐selectin; (G) IOD of P‐selectin. Scale bars, 100 μm. * P

    Article Snippet: 2.16 Experimental grouping and processing of the in vitro experiments mDCs was randomly divided into five groups, each group had three time points of 24, 48 and 72 hours, with three wells in each group. (a) C group: control group, mDCs was added to the complete medium of RPMI l640; (b) H group: HMGB1 (Ab215008; Abcam) was added to the complete medium containing mDCs (Ab215008, Abcam) (1 μg/mL); (c) H‐H‐Ig group: HMGB1 (1ug/mL) was added followed by HMGB1 specific neutralizing antibody (ST326052233; Shino‐Test, Tokyo, Japan) (the final concentration was 1 μg/mL) was added 30 minutes later; (d) H‐TLR4‐A group: HMGB1(1ug/ml) was added followed by TLR4 antagonists (A3850; ApexBio, Boston, MA) (the final concentration was 1 μg/mL) 30 minutes later; (e) H‐Ig group: HMGB1 (1ug/mL) was added followed by control IgG antibody (ST326058471; Shino‐Test) (the final concentration was 1 μg/mL) 30 minutes later.

    Techniques: Migration, Activation Assay, Immunofluorescence, Staining, Fluorescence, Flow Cytometry, Cytometry, Immunohistochemistry

    High‐mobility group protein box‐1 (HMGB1) antibody attenuates myocardial injury through mediating the role of dendritic cells (DCs) regulated by the HMGB1‐TLR4 signalling pathway. A, Comparison of myocardial infarct size among all groups (n = 6 for each group). Anti‐HMGB1 treatment reduces myocardial IR injury. Representative images of the infract area (INF: white), area at risk (AAR: red and white), and normal area (blue). B, Quantitative analysis of infarct size and the INF/AAR ratio (IAR, %). Comparison of concentrations of serum CK‐MB, cTnI, HMGB1, and IL‐6, IL‐8, TNF‐α, IL‐12 among all groups (n = 10 for each group). Blood was collected right after the IR procedure was completed. CK‐MB, cTnI, HMGB1, and IL‐6, IL‐8, TNF‐α, IL‐12 were measured as described in the Section 2 . C, CK‐MB. D, CTnI. E, HMGB1. F, Inflammatory factors (IL‐6, IL‐8, TNF‐α, IL‐12). Comparison of protein and mRNA levels of HMGB1, TLR4, NF‐κB of cardiac tissues from rats of different groups (n = 10 for each group). Samples were collected right after IR procedure was completed. G, Western blot analysis of HMGB1 protein expression; H, Western blot analysis of TLR4 protein expression; I, Western blot analysis of NF‐κB protein expression; (J) relative expression of HMGB1 mRNA; (K) relative expression of TLR4 mRNA; (L) relative expression of NF‐κB mRNA. * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: The role of dendritic cells regulated by HMGB1/TLR4 signalling pathway in myocardial ischaemia reperfusion injury, et al. The role of dendritic cells regulated by HMGB1/TLR4 signalling pathway in myocardial ischaemia reperfusion injury

    doi: 10.1111/jcmm.14192

    Figure Lengend Snippet: High‐mobility group protein box‐1 (HMGB1) antibody attenuates myocardial injury through mediating the role of dendritic cells (DCs) regulated by the HMGB1‐TLR4 signalling pathway. A, Comparison of myocardial infarct size among all groups (n = 6 for each group). Anti‐HMGB1 treatment reduces myocardial IR injury. Representative images of the infract area (INF: white), area at risk (AAR: red and white), and normal area (blue). B, Quantitative analysis of infarct size and the INF/AAR ratio (IAR, %). Comparison of concentrations of serum CK‐MB, cTnI, HMGB1, and IL‐6, IL‐8, TNF‐α, IL‐12 among all groups (n = 10 for each group). Blood was collected right after the IR procedure was completed. CK‐MB, cTnI, HMGB1, and IL‐6, IL‐8, TNF‐α, IL‐12 were measured as described in the Section 2 . C, CK‐MB. D, CTnI. E, HMGB1. F, Inflammatory factors (IL‐6, IL‐8, TNF‐α, IL‐12). Comparison of protein and mRNA levels of HMGB1, TLR4, NF‐κB of cardiac tissues from rats of different groups (n = 10 for each group). Samples were collected right after IR procedure was completed. G, Western blot analysis of HMGB1 protein expression; H, Western blot analysis of TLR4 protein expression; I, Western blot analysis of NF‐κB protein expression; (J) relative expression of HMGB1 mRNA; (K) relative expression of TLR4 mRNA; (L) relative expression of NF‐κB mRNA. * P

    Article Snippet: 2.16 Experimental grouping and processing of the in vitro experiments mDCs was randomly divided into five groups, each group had three time points of 24, 48 and 72 hours, with three wells in each group. (a) C group: control group, mDCs was added to the complete medium of RPMI l640; (b) H group: HMGB1 (Ab215008; Abcam) was added to the complete medium containing mDCs (Ab215008, Abcam) (1 μg/mL); (c) H‐H‐Ig group: HMGB1 (1ug/mL) was added followed by HMGB1 specific neutralizing antibody (ST326052233; Shino‐Test, Tokyo, Japan) (the final concentration was 1 μg/mL) was added 30 minutes later; (d) H‐TLR4‐A group: HMGB1(1ug/ml) was added followed by TLR4 antagonists (A3850; ApexBio, Boston, MA) (the final concentration was 1 μg/mL) 30 minutes later; (e) H‐Ig group: HMGB1 (1ug/mL) was added followed by control IgG antibody (ST326058471; Shino‐Test) (the final concentration was 1 μg/mL) 30 minutes later.

    Techniques: Western Blot, Expressing

    Doppler echocardiography. (A‐D) The M‐mode echocardiograms of the short‐axis midventricular view of different groups. (E‐G), The changes of left ventricular internal dimension in diastole (LVIDd), left ventricular internal dimension in systole (LVIDs) and left ventricular ejection fraction (LVEF) of different groups (n = 6 for each group). Anti‐HMGB1 treatment might improve heart function. * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: The role of dendritic cells regulated by HMGB1/TLR4 signalling pathway in myocardial ischaemia reperfusion injury, et al. The role of dendritic cells regulated by HMGB1/TLR4 signalling pathway in myocardial ischaemia reperfusion injury

    doi: 10.1111/jcmm.14192

    Figure Lengend Snippet: Doppler echocardiography. (A‐D) The M‐mode echocardiograms of the short‐axis midventricular view of different groups. (E‐G), The changes of left ventricular internal dimension in diastole (LVIDd), left ventricular internal dimension in systole (LVIDs) and left ventricular ejection fraction (LVEF) of different groups (n = 6 for each group). Anti‐HMGB1 treatment might improve heart function. * P

    Article Snippet: 2.16 Experimental grouping and processing of the in vitro experiments mDCs was randomly divided into five groups, each group had three time points of 24, 48 and 72 hours, with three wells in each group. (a) C group: control group, mDCs was added to the complete medium of RPMI l640; (b) H group: HMGB1 (Ab215008; Abcam) was added to the complete medium containing mDCs (Ab215008, Abcam) (1 μg/mL); (c) H‐H‐Ig group: HMGB1 (1ug/mL) was added followed by HMGB1 specific neutralizing antibody (ST326052233; Shino‐Test, Tokyo, Japan) (the final concentration was 1 μg/mL) was added 30 minutes later; (d) H‐TLR4‐A group: HMGB1(1ug/ml) was added followed by TLR4 antagonists (A3850; ApexBio, Boston, MA) (the final concentration was 1 μg/mL) 30 minutes later; (e) H‐Ig group: HMGB1 (1ug/mL) was added followed by control IgG antibody (ST326058471; Shino‐Test) (the final concentration was 1 μg/mL) 30 minutes later.

    Techniques:

    HMGB1 aggravated macrophage inflammatory response. (a-b) RAW264.7 cells were stimulated with ALD-DNA (0, 25, 50 μ g/mL) for 24 h, levels of HMGB1 in the supernatants of RAW264.7 cells were analyzed by ELISA (a) and western blot analysis (b). Data are means ± SD of three independent experiments. (c-d) The efficiency of HMGB1 overexpression (c) and knockdown (d) was monitored by representative immunoblot of three independent experiments in ALD-DNA-stimulated RAW264.7 cells. (e-f) RAW264.7 cells were transfected with pHMGB1 or vector. 72 h after transfection, RAW264.7 cells were stimulated with PBS, UnALD-DNA or ALD-DNA (50 μ g/mL) followed by analyzing the levels of TNF- α (e) and IL-6 (f) in the culture supernatants of RAW264.7 cells. Data are means ± SD of three independent experiments. (g-h) RAW264.7 cells were transfected with control siRNA (200 nM) or HMGB1 siRNA (siHMGB1, 200 nM). After 72 h RAW264.7 cells were stimulated with PBS, UnALD-DNA or ALD-DNA (50 μ g/mL). ELISA assay was used to analyze the levels of TNF- α (g) and IL-6 (h) in the culture supernatants of RAW264.7 cells. Data are means ± SD of three independent experiments. (i-j) CD11b + /F4/80 high renal macrophages were sorted from nephritic single-cell suspensions from (i) pHMGB1- or empty vector-treated, (j) glycyrrhizin- or PBS-treated SLE mice by flow cytometry. Macrophages (2 × 10 5 /mL) were stimulated with ALD-DNA (50 μ g/mL) for 24 h. The supernatants were collected and assayed for the TNF- α and IL-6 concentrations using ELISA. Data are means ± SD from 8 mice in each group. ∗ P

    Journal: Journal of Immunology Research

    Article Title: HMGB1 Promotes Systemic Lupus Erythematosus by Enhancing Macrophage Inflammatory Response

    doi: 10.1155/2015/946748

    Figure Lengend Snippet: HMGB1 aggravated macrophage inflammatory response. (a-b) RAW264.7 cells were stimulated with ALD-DNA (0, 25, 50 μ g/mL) for 24 h, levels of HMGB1 in the supernatants of RAW264.7 cells were analyzed by ELISA (a) and western blot analysis (b). Data are means ± SD of three independent experiments. (c-d) The efficiency of HMGB1 overexpression (c) and knockdown (d) was monitored by representative immunoblot of three independent experiments in ALD-DNA-stimulated RAW264.7 cells. (e-f) RAW264.7 cells were transfected with pHMGB1 or vector. 72 h after transfection, RAW264.7 cells were stimulated with PBS, UnALD-DNA or ALD-DNA (50 μ g/mL) followed by analyzing the levels of TNF- α (e) and IL-6 (f) in the culture supernatants of RAW264.7 cells. Data are means ± SD of three independent experiments. (g-h) RAW264.7 cells were transfected with control siRNA (200 nM) or HMGB1 siRNA (siHMGB1, 200 nM). After 72 h RAW264.7 cells were stimulated with PBS, UnALD-DNA or ALD-DNA (50 μ g/mL). ELISA assay was used to analyze the levels of TNF- α (g) and IL-6 (h) in the culture supernatants of RAW264.7 cells. Data are means ± SD of three independent experiments. (i-j) CD11b + /F4/80 high renal macrophages were sorted from nephritic single-cell suspensions from (i) pHMGB1- or empty vector-treated, (j) glycyrrhizin- or PBS-treated SLE mice by flow cytometry. Macrophages (2 × 10 5 /mL) were stimulated with ALD-DNA (50 μ g/mL) for 24 h. The supernatants were collected and assayed for the TNF- α and IL-6 concentrations using ELISA. Data are means ± SD from 8 mice in each group. ∗ P

    Article Snippet: To further confirm the effect of HMGB1 on the progression of SLE, we inhibited the function of HMGB1 in vivo by injecting BALB/c mice intramuscularly with glycyrrhizin which has been demonstrated to be the blocker of HMGB1 [ – ].

    Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Over Expression, Transfection, Plasmid Preparation, Mouse Assay, Flow Cytometry, Cytometry

    The HMGB1 levels were associated with proinflammatory cytokines in SLE patients. (a-b) TNF- α (a) and IL-6 (b) concentrations in sera from SLE patients and HC were detected by ELISA. The scatter-plot represented the TNF- α and IL-6 levels by ELISA analysis. Each symbol represents one SLE patient. Horizontal lines represent the median. Data represent the average from experiments performed in triplicates for each patient. (c-d) Correlation analyses were presented between HMGB1 and TNF- α levels (c), HMGB1 and IL-6 levels (d). Pearson correlation analysis was used in the correlation analysis. ∗ P

    Journal: Journal of Immunology Research

    Article Title: HMGB1 Promotes Systemic Lupus Erythematosus by Enhancing Macrophage Inflammatory Response

    doi: 10.1155/2015/946748

    Figure Lengend Snippet: The HMGB1 levels were associated with proinflammatory cytokines in SLE patients. (a-b) TNF- α (a) and IL-6 (b) concentrations in sera from SLE patients and HC were detected by ELISA. The scatter-plot represented the TNF- α and IL-6 levels by ELISA analysis. Each symbol represents one SLE patient. Horizontal lines represent the median. Data represent the average from experiments performed in triplicates for each patient. (c-d) Correlation analyses were presented between HMGB1 and TNF- α levels (c), HMGB1 and IL-6 levels (d). Pearson correlation analysis was used in the correlation analysis. ∗ P

    Article Snippet: To further confirm the effect of HMGB1 on the progression of SLE, we inhibited the function of HMGB1 in vivo by injecting BALB/c mice intramuscularly with glycyrrhizin which has been demonstrated to be the blocker of HMGB1 [ – ].

    Techniques: Enzyme-linked Immunosorbent Assay

    HMGB1 overexpression could promote the severity of SLE. BALB/c mice were administrated intramuscularly with 100 μ g pHMGB1 or empty-vector per mouse. 72 h following injection, mice were then injected subcutaneously with ALD-DNA (50 μ g/mouse) for total 3 times in 4 weeks. (a) The dynamics of serum HMGB1 levels were determined by ELISA every 2 weeks after initial injection. Data are means ± SD from 8 mice in each group. (b) Nephritic pathology was evaluated by H E staining of renal tissues. Images (magnification ×200) are representative of at least 8 mice in each group. (c) The kidney score was assessed using paraffin sections stained with H E in (b). ( n = 8). (d) Serum anti-dsDNA antibody levels were measured by ELISA every 2 weeks after initial injection. Data are means ± SD from 8 mice in each group. (e) Urine protein levels of the mice were assessed by BCA method every 2 weeks. Data are means ± SD from 8 mice in each group. ∗ P

    Journal: Journal of Immunology Research

    Article Title: HMGB1 Promotes Systemic Lupus Erythematosus by Enhancing Macrophage Inflammatory Response

    doi: 10.1155/2015/946748

    Figure Lengend Snippet: HMGB1 overexpression could promote the severity of SLE. BALB/c mice were administrated intramuscularly with 100 μ g pHMGB1 or empty-vector per mouse. 72 h following injection, mice were then injected subcutaneously with ALD-DNA (50 μ g/mouse) for total 3 times in 4 weeks. (a) The dynamics of serum HMGB1 levels were determined by ELISA every 2 weeks after initial injection. Data are means ± SD from 8 mice in each group. (b) Nephritic pathology was evaluated by H E staining of renal tissues. Images (magnification ×200) are representative of at least 8 mice in each group. (c) The kidney score was assessed using paraffin sections stained with H E in (b). ( n = 8). (d) Serum anti-dsDNA antibody levels were measured by ELISA every 2 weeks after initial injection. Data are means ± SD from 8 mice in each group. (e) Urine protein levels of the mice were assessed by BCA method every 2 weeks. Data are means ± SD from 8 mice in each group. ∗ P

    Article Snippet: To further confirm the effect of HMGB1 on the progression of SLE, we inhibited the function of HMGB1 in vivo by injecting BALB/c mice intramuscularly with glycyrrhizin which has been demonstrated to be the blocker of HMGB1 [ – ].

    Techniques: Over Expression, Mouse Assay, Plasmid Preparation, Injection, Enzyme-linked Immunosorbent Assay, Staining, BIA-KA

    Inhibition of HMGB1 function could ameliorate the severity of SLE. BALB/c mice were administrated intramuscularly injected with glycyrrhizin (0.5 mg/mouse) or PBS to inhibit HMGB1 function. 72 h after injection, mice were then injected subcutaneously with ALD-DNA (50 μ g/mouse) for total 3 times in 4 weeks. (a) The dynamics of serum HMGB1 levels were determined by ELISA every 2 weeks after initial injection. Data are means ± SD from 8 mice in each group. (b) Nephritic pathology was evaluated by H E staining of renal tissues. Images (magnification ×200) are representative of at least 8 mice in each group. (c) The kidney score was assessed using paraffin sections stained with H E in (b). ( n = 8). (d) Serum anti-dsDNA antibody levels were measured by ELISA every 2 weeks after initial injection. Data are means ± SD from 8 mice in each group. (e) Urine protein levels of the mice were assessed by BCA method every 2 weeks. Data are means ± SD from 8 mice in each group. ∗ P

    Journal: Journal of Immunology Research

    Article Title: HMGB1 Promotes Systemic Lupus Erythematosus by Enhancing Macrophage Inflammatory Response

    doi: 10.1155/2015/946748

    Figure Lengend Snippet: Inhibition of HMGB1 function could ameliorate the severity of SLE. BALB/c mice were administrated intramuscularly injected with glycyrrhizin (0.5 mg/mouse) or PBS to inhibit HMGB1 function. 72 h after injection, mice were then injected subcutaneously with ALD-DNA (50 μ g/mouse) for total 3 times in 4 weeks. (a) The dynamics of serum HMGB1 levels were determined by ELISA every 2 weeks after initial injection. Data are means ± SD from 8 mice in each group. (b) Nephritic pathology was evaluated by H E staining of renal tissues. Images (magnification ×200) are representative of at least 8 mice in each group. (c) The kidney score was assessed using paraffin sections stained with H E in (b). ( n = 8). (d) Serum anti-dsDNA antibody levels were measured by ELISA every 2 weeks after initial injection. Data are means ± SD from 8 mice in each group. (e) Urine protein levels of the mice were assessed by BCA method every 2 weeks. Data are means ± SD from 8 mice in each group. ∗ P

    Article Snippet: To further confirm the effect of HMGB1 on the progression of SLE, we inhibited the function of HMGB1 in vivo by injecting BALB/c mice intramuscularly with glycyrrhizin which has been demonstrated to be the blocker of HMGB1 [ – ].

    Techniques: Inhibition, Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay, Staining, BIA-KA

    HMGB1 levels were elevated and correlated with SLE disease activity in both clinical patients and murine model. (a) Serum HMGB1 levels were detected by ELISA in SLE patients and HC. (b) HMGB1 levels were measured by ELISA in renal active or inactive patients. The scatter-plot represented the HMGB1 levels by ELISA. Each symbol represents one SLE patient. Horizontal lines represent the median. Data represent the average from experiments performed in triplicates for each patient. (c) Correlation analysis was performed between HMGB1 levels and SLEDAI. (d) Correlation analysis was performed between HMGB1 and anti-dsDNA antibody levels. Pearson correlation analysis was used in the correlation analysis. (e) The expression of HMGB1 was analyzed by western blot in PBMCs from SLE patients (S) and healthy controls (H). Representative western blot bands from 4 patients with SLE and 4 HC were presented. Data were representative of results obtained in three independent experiments. (f) Serum HMGB1 levels were measured by ELISA every 2 weeks after initial injection. Data are means ± SD from 8 mice in each group. (g) The correlation between serum HMGB1 levels and kidney score was carried out in SLE mice. (h) The correlation between serum HMGB1 and anti-dsDNA antibody levels was carried out in SLE mice. (i) The correlation between serum HMGB1 and urine protein levels was carried out in SLE mice. Pearson correlation analysis was used to carry out the correlation study. Each symbol indicates an individual mouse ( n = 19). ∗ P

    Journal: Journal of Immunology Research

    Article Title: HMGB1 Promotes Systemic Lupus Erythematosus by Enhancing Macrophage Inflammatory Response

    doi: 10.1155/2015/946748

    Figure Lengend Snippet: HMGB1 levels were elevated and correlated with SLE disease activity in both clinical patients and murine model. (a) Serum HMGB1 levels were detected by ELISA in SLE patients and HC. (b) HMGB1 levels were measured by ELISA in renal active or inactive patients. The scatter-plot represented the HMGB1 levels by ELISA. Each symbol represents one SLE patient. Horizontal lines represent the median. Data represent the average from experiments performed in triplicates for each patient. (c) Correlation analysis was performed between HMGB1 levels and SLEDAI. (d) Correlation analysis was performed between HMGB1 and anti-dsDNA antibody levels. Pearson correlation analysis was used in the correlation analysis. (e) The expression of HMGB1 was analyzed by western blot in PBMCs from SLE patients (S) and healthy controls (H). Representative western blot bands from 4 patients with SLE and 4 HC were presented. Data were representative of results obtained in three independent experiments. (f) Serum HMGB1 levels were measured by ELISA every 2 weeks after initial injection. Data are means ± SD from 8 mice in each group. (g) The correlation between serum HMGB1 levels and kidney score was carried out in SLE mice. (h) The correlation between serum HMGB1 and anti-dsDNA antibody levels was carried out in SLE mice. (i) The correlation between serum HMGB1 and urine protein levels was carried out in SLE mice. Pearson correlation analysis was used to carry out the correlation study. Each symbol indicates an individual mouse ( n = 19). ∗ P

    Article Snippet: To further confirm the effect of HMGB1 on the progression of SLE, we inhibited the function of HMGB1 in vivo by injecting BALB/c mice intramuscularly with glycyrrhizin which has been demonstrated to be the blocker of HMGB1 [ – ].

    Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Injection, Mouse Assay

    HMGB1-enhanced macrophage inflammatory response induced by ALD-DNA might be dependent on RAGE but not on TLR2 and TLR4. (a-b) RAW264.7 cells were transfected with pHMGB1, and then stimulated with ALD-DNA (50 μ g/mL) in the presence of OxPAPC (30 μ g/mL) or RAGE-Fc (10 μ g/mL) for 24 h. The supernatants were collected and assayed for the concentrations of TNF- α (a) and IL-6 (b) using ELISA. (c) Representative immunoblot of three independent experiments has shown the efficiency of RAGE knockdown. (d-e) RAW264.7 cells transfected with siRAGE and pHMGB1 were stimulated with 50 μ g/mL of ALD-DNA for 24 h. The supernatants were collected and assayed for the concentrations TNF- α (d) and IL-6 (e) using ELISA. ∗ P

    Journal: Journal of Immunology Research

    Article Title: HMGB1 Promotes Systemic Lupus Erythematosus by Enhancing Macrophage Inflammatory Response

    doi: 10.1155/2015/946748

    Figure Lengend Snippet: HMGB1-enhanced macrophage inflammatory response induced by ALD-DNA might be dependent on RAGE but not on TLR2 and TLR4. (a-b) RAW264.7 cells were transfected with pHMGB1, and then stimulated with ALD-DNA (50 μ g/mL) in the presence of OxPAPC (30 μ g/mL) or RAGE-Fc (10 μ g/mL) for 24 h. The supernatants were collected and assayed for the concentrations of TNF- α (a) and IL-6 (b) using ELISA. (c) Representative immunoblot of three independent experiments has shown the efficiency of RAGE knockdown. (d-e) RAW264.7 cells transfected with siRAGE and pHMGB1 were stimulated with 50 μ g/mL of ALD-DNA for 24 h. The supernatants were collected and assayed for the concentrations TNF- α (d) and IL-6 (e) using ELISA. ∗ P

    Article Snippet: To further confirm the effect of HMGB1 on the progression of SLE, we inhibited the function of HMGB1 in vivo by injecting BALB/c mice intramuscularly with glycyrrhizin which has been demonstrated to be the blocker of HMGB1 [ – ].

    Techniques: Transfection, Enzyme-linked Immunosorbent Assay

    The relative expression of HMGB1 and HMGN2 mRNA in gingival tissues from the patients in CP, G-AgP and healthy groups. A. Values were presented as mean ± SD for at least three independent experiments performed in triplicate. The expressions of HMGB1 and HMGN2 in control group were normalized in 1.0. B. Representative RT-PCR images for HMGB1 and HMGN2 in four groups.

    Journal: Brazilian Journal of Microbiology

    Article Title: Expression of HMGB1 and HMGN2 in gingival tissues, GCF and PICF of periodontitis patients and peri-implantitis

    doi: 10.1590/S1517-838220110003000047

    Figure Lengend Snippet: The relative expression of HMGB1 and HMGN2 mRNA in gingival tissues from the patients in CP, G-AgP and healthy groups. A. Values were presented as mean ± SD for at least three independent experiments performed in triplicate. The expressions of HMGB1 and HMGN2 in control group were normalized in 1.0. B. Representative RT-PCR images for HMGB1 and HMGN2 in four groups.

    Article Snippet: The level of HMGB1 was the highest in the CP group.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

    Representative Western blotting images for HMGB1 and HMGN2 expression in GCF from CP, G-AgP and control groups and in PICF in PI and normal group. β-actin served as the loading control.

    Journal: Brazilian Journal of Microbiology

    Article Title: Expression of HMGB1 and HMGN2 in gingival tissues, GCF and PICF of periodontitis patients and peri-implantitis

    doi: 10.1590/S1517-838220110003000047

    Figure Lengend Snippet: Representative Western blotting images for HMGB1 and HMGN2 expression in GCF from CP, G-AgP and control groups and in PICF in PI and normal group. β-actin served as the loading control.

    Article Snippet: The level of HMGB1 was the highest in the CP group.

    Techniques: Western Blot, Expressing

    Suppression of systemic inflammatory aggravation in PSI animals by blocking HMGB1 function. a Animals were administered HMGB1 A box (5 μg/kg) or HPep1 (5 μg/kg) intranasally 3 h prior to LPS administration (100 μg/kg, i.p.) at 24 h post-MCAO. b , c Serum HMGB1 levels were examined at 2 or 4 days post-MCAO for MCAO or PSI animals with or without A box or HPep1 treatment at 21 h post-MCAO by immunoblotting. d , e Levels of TNF-α and IL-1β in the serum were measured in MCAO, MCAO+LPS, MCAO+LPS+A box (5 μg/kg), or MCAO+LPS+HPep1 (5 μg/kg) group at 2 days post-MCAO by ELISA. Results are presented as means ± SEMs ( n = 4). Sham or S sham-operated rats, LPS LPS-treated rats, MCAO treatment-naive MCAO rats, MCAO+LPS LPS-treated MCAO rats, MCAO+LPS+A box A box-treated MCAO+LPS rats, MCAO+LPS+HPep1 HPep1-treated MCAO+LPS rats. ** p

    Journal: Cell Death & Disease

    Article Title: Alarmin HMGB1 induces systemic and brain inflammatory exacerbation in post-stroke infection rat model

    doi: 10.1038/s41419-018-0438-8

    Figure Lengend Snippet: Suppression of systemic inflammatory aggravation in PSI animals by blocking HMGB1 function. a Animals were administered HMGB1 A box (5 μg/kg) or HPep1 (5 μg/kg) intranasally 3 h prior to LPS administration (100 μg/kg, i.p.) at 24 h post-MCAO. b , c Serum HMGB1 levels were examined at 2 or 4 days post-MCAO for MCAO or PSI animals with or without A box or HPep1 treatment at 21 h post-MCAO by immunoblotting. d , e Levels of TNF-α and IL-1β in the serum were measured in MCAO, MCAO+LPS, MCAO+LPS+A box (5 μg/kg), or MCAO+LPS+HPep1 (5 μg/kg) group at 2 days post-MCAO by ELISA. Results are presented as means ± SEMs ( n = 4). Sham or S sham-operated rats, LPS LPS-treated rats, MCAO treatment-naive MCAO rats, MCAO+LPS LPS-treated MCAO rats, MCAO+LPS+A box A box-treated MCAO+LPS rats, MCAO+LPS+HPep1 HPep1-treated MCAO+LPS rats. ** p

    Article Snippet: After blocking membranes with 5% non-fat milk for 1 h, they were incubated with primary antibodies for anti-HMGB1 (1:2000; Abcam, Cambridge, UK; ab67281), anti-albumin (1:5000; Santa Cruz Biotechnology, Santa Cruz, CA, USA; SC-374670), and anti-α-tubulin (1:5000; Cell Signaling, Danvers, MA, USA; #2144) overnight at 4 °C.

    Techniques: Blocking Assay, Enzyme-linked Immunosorbent Assay

    Enhanced binding between LPS and HMGB1 in sera of LPS-injected MCAO animals and inhibition of LPS-HMGB1 binding by blocking HMGB1. Binding between HMGB1 and LPS was examined by co-immunoprecipitation-immunoblotting assay. a Serum was prepared from MCAO or PSI animals at 1 or 2 days post-MCAO and precipitated with anti-LPS antibody, and amounts of HMGB1 were determined by immunoblotting using anti-HMGB1 antibody. Amounts of HMGB1, LPS, or serum albumin before immunoprecipitations are presented as input controls. b Serum was prepared from MCAO or PSI animals at 2 days post-MCAO after pre-administrating HMGB1 A box (5 μg/kg) or HPep1 (5 μg/kg) at 21 h post-MCAO, and the same co-immunoprecipitation experiments were carried out. c , d Co-immunoprecipitation-immunoblotting assay was carried out using brain tissues prepared from the cortical penumbra of MCAO or PSI animals at 2 days post-MCAO. Results are presented as mean ± SEM ( n = 4). Sham sham-operated rats. ** p

    Journal: Cell Death & Disease

    Article Title: Alarmin HMGB1 induces systemic and brain inflammatory exacerbation in post-stroke infection rat model

    doi: 10.1038/s41419-018-0438-8

    Figure Lengend Snippet: Enhanced binding between LPS and HMGB1 in sera of LPS-injected MCAO animals and inhibition of LPS-HMGB1 binding by blocking HMGB1. Binding between HMGB1 and LPS was examined by co-immunoprecipitation-immunoblotting assay. a Serum was prepared from MCAO or PSI animals at 1 or 2 days post-MCAO and precipitated with anti-LPS antibody, and amounts of HMGB1 were determined by immunoblotting using anti-HMGB1 antibody. Amounts of HMGB1, LPS, or serum albumin before immunoprecipitations are presented as input controls. b Serum was prepared from MCAO or PSI animals at 2 days post-MCAO after pre-administrating HMGB1 A box (5 μg/kg) or HPep1 (5 μg/kg) at 21 h post-MCAO, and the same co-immunoprecipitation experiments were carried out. c , d Co-immunoprecipitation-immunoblotting assay was carried out using brain tissues prepared from the cortical penumbra of MCAO or PSI animals at 2 days post-MCAO. Results are presented as mean ± SEM ( n = 4). Sham sham-operated rats. ** p

    Article Snippet: After blocking membranes with 5% non-fat milk for 1 h, they were incubated with primary antibodies for anti-HMGB1 (1:2000; Abcam, Cambridge, UK; ab67281), anti-albumin (1:5000; Santa Cruz Biotechnology, Santa Cruz, CA, USA; SC-374670), and anti-α-tubulin (1:5000; Cell Signaling, Danvers, MA, USA; #2144) overnight at 4 °C.

    Techniques: Binding Assay, Injection, Inhibition, Blocking Assay, Immunoprecipitation

    Stroke severity and HMGB1 levels in the serum and CSF. MCA of Sprague–Dawley rats were occluded for 30, 60, 90, or 120 min and mean infarct volumes were assessed at 2 days post-MCAO by TTC staining. Representative images of infarctions in coronal brain sections are presented ( a ) and mean infarction volumes are presented as means ± SEMs ( n = 6–8) ( b ). Representative images of infarctions in coronal brain sections at 1 day post-MCAO are presented ( c ). Levels of HMGB1 in the serum ( d , e ), CSF ( d , f ), and in cortex core and penumbra of the ischemic hemisphere indicated in c ( g – i ) at 1 day post-MCAO (30, 60, 90, or 120 min occlusion) were examined by immunoblotting. Sham or S sham-operated rats. ¢¢ p

    Journal: Cell Death & Disease

    Article Title: Alarmin HMGB1 induces systemic and brain inflammatory exacerbation in post-stroke infection rat model

    doi: 10.1038/s41419-018-0438-8

    Figure Lengend Snippet: Stroke severity and HMGB1 levels in the serum and CSF. MCA of Sprague–Dawley rats were occluded for 30, 60, 90, or 120 min and mean infarct volumes were assessed at 2 days post-MCAO by TTC staining. Representative images of infarctions in coronal brain sections are presented ( a ) and mean infarction volumes are presented as means ± SEMs ( n = 6–8) ( b ). Representative images of infarctions in coronal brain sections at 1 day post-MCAO are presented ( c ). Levels of HMGB1 in the serum ( d , e ), CSF ( d , f ), and in cortex core and penumbra of the ischemic hemisphere indicated in c ( g – i ) at 1 day post-MCAO (30, 60, 90, or 120 min occlusion) were examined by immunoblotting. Sham or S sham-operated rats. ¢¢ p

    Article Snippet: After blocking membranes with 5% non-fat milk for 1 h, they were incubated with primary antibodies for anti-HMGB1 (1:2000; Abcam, Cambridge, UK; ab67281), anti-albumin (1:5000; Santa Cruz Biotechnology, Santa Cruz, CA, USA; SC-374670), and anti-α-tubulin (1:5000; Cell Signaling, Danvers, MA, USA; #2144) overnight at 4 °C.

    Techniques: Staining

    HMGB1 surge in the serum of PSI animals. Serum HMGB1 levels were examined for the MCAO or PSI (100 μg/kg of LPS injected at 24 h post-MCAO) animals at 1, 2, 4, 7, and 10 days post-MCAO ( a , b ) or for the MCAO+LPS+LPS-RS group ( c , d ). e , f Relative levels of disulfide-HMGB1 or reduced-HMGB1 were examined four independent experiments and representative pictures are presented ( e ) and results are presented as the mean ± SEM ( f ). Sham sham-operated rats, MCAO treatment-naive MCAO rats ( n = 5), MCAO+LPS LPS-treated MCAO rats ( n = 5), MCAO+LPS+LPS-RS LPS-treated and LPS-RS-treated MCAO rats ( n = 4). * p

    Journal: Cell Death & Disease

    Article Title: Alarmin HMGB1 induces systemic and brain inflammatory exacerbation in post-stroke infection rat model

    doi: 10.1038/s41419-018-0438-8

    Figure Lengend Snippet: HMGB1 surge in the serum of PSI animals. Serum HMGB1 levels were examined for the MCAO or PSI (100 μg/kg of LPS injected at 24 h post-MCAO) animals at 1, 2, 4, 7, and 10 days post-MCAO ( a , b ) or for the MCAO+LPS+LPS-RS group ( c , d ). e , f Relative levels of disulfide-HMGB1 or reduced-HMGB1 were examined four independent experiments and representative pictures are presented ( e ) and results are presented as the mean ± SEM ( f ). Sham sham-operated rats, MCAO treatment-naive MCAO rats ( n = 5), MCAO+LPS LPS-treated MCAO rats ( n = 5), MCAO+LPS+LPS-RS LPS-treated and LPS-RS-treated MCAO rats ( n = 4). * p

    Article Snippet: After blocking membranes with 5% non-fat milk for 1 h, they were incubated with primary antibodies for anti-HMGB1 (1:2000; Abcam, Cambridge, UK; ab67281), anti-albumin (1:5000; Santa Cruz Biotechnology, Santa Cruz, CA, USA; SC-374670), and anti-α-tubulin (1:5000; Cell Signaling, Danvers, MA, USA; #2144) overnight at 4 °C.

    Techniques: Injection

    The Evaluation of cytosolic and serum high mobility group box 1 (HMGB1) in 5-FU-induced oral mucositis. ( A ) Immunohistochemical analysis of cytosolic HMGB1 expression in the oral mucosa. The number of HMGB1-positive cells per field in the lesion have been count. * p

    Journal: International Journal of Molecular Sciences

    Article Title: Anthocyanins Extracted from Oryza sativa L. Prevent Fluorouracil-Induced Nuclear Factor-κB Activation in Oral Mucositis: In Vitro and In Vivo Studies

    doi: 10.3390/ijms19102981

    Figure Lengend Snippet: The Evaluation of cytosolic and serum high mobility group box 1 (HMGB1) in 5-FU-induced oral mucositis. ( A ) Immunohistochemical analysis of cytosolic HMGB1 expression in the oral mucosa. The number of HMGB1-positive cells per field in the lesion have been count. * p

    Article Snippet: HMGB1 Ab and cell fractionation kit were obstained from Abcam (Cambridge, MA, USA).

    Techniques: Immunohistochemistry, Expressing

    Effect of HMGB1 and IL-1β on the levels of MMP released into the medium by osteoarthritic synoviocytes . (a) Matrix metalloproteinase (MMP)-1, (b) MMP-3 and (c) MMP-13 protein levels in the medium. Cells were stimulated with IL-1b (10 ng/ml) for 24 hours in the presence or absence of high mobility group box 1 (HMGB1) at 15 and 25 ng/ml. MMP protein was measured by ELISA in supernatants. Data are expressed as mean ± standard error of the mean. Duplicate samples from six to eight patients were used. ++ P

    Journal: Arthritis Research & Therapy

    Article Title: High mobility group box 1 potentiates the pro-inflammatory effects of interleukin-1? in osteoarthritic synoviocytes

    doi: 10.1186/ar3124

    Figure Lengend Snippet: Effect of HMGB1 and IL-1β on the levels of MMP released into the medium by osteoarthritic synoviocytes . (a) Matrix metalloproteinase (MMP)-1, (b) MMP-3 and (c) MMP-13 protein levels in the medium. Cells were stimulated with IL-1b (10 ng/ml) for 24 hours in the presence or absence of high mobility group box 1 (HMGB1) at 15 and 25 ng/ml. MMP protein was measured by ELISA in supernatants. Data are expressed as mean ± standard error of the mean. Duplicate samples from six to eight patients were used. ++ P

    Article Snippet: Western blot analysis After stimulation for five minutes with IL-1β (10 ng/ml), HMGB1 at 15 and 25 ng/ml or IL-1β+HMGB1, synoviocytes were lysed in 100 μl of buffer (1% Triton X-100, 1% deoxycholic acid, 20 mM NaCl and 25 mM Tris, pH 7.4) and centrifuged at 4°C for 15 minutes at 10,000g .

    Techniques: Enzyme-linked Immunosorbent Assay

    Effect of HMGB1 and IL-1β on NF-κB activation . Transient transfection was performed with the reporter construct nuclear factor (NF)-κB-luc and the internal control pRL-TK, as indicated in Materials and Methods. Cells were treated for 24 hours with high mobility group box 1 (HMGB1) at 25 ng/ml in the absence or presence of IL-1β (10 ng/ml). Firefly luciferase activity was normalized to Renilla luciferase activity. Data are expressed as mean ± standard error of the mean. Duplicate samples from six patients were used. ++ P

    Journal: Arthritis Research & Therapy

    Article Title: High mobility group box 1 potentiates the pro-inflammatory effects of interleukin-1? in osteoarthritic synoviocytes

    doi: 10.1186/ar3124

    Figure Lengend Snippet: Effect of HMGB1 and IL-1β on NF-κB activation . Transient transfection was performed with the reporter construct nuclear factor (NF)-κB-luc and the internal control pRL-TK, as indicated in Materials and Methods. Cells were treated for 24 hours with high mobility group box 1 (HMGB1) at 25 ng/ml in the absence or presence of IL-1β (10 ng/ml). Firefly luciferase activity was normalized to Renilla luciferase activity. Data are expressed as mean ± standard error of the mean. Duplicate samples from six patients were used. ++ P

    Article Snippet: Western blot analysis After stimulation for five minutes with IL-1β (10 ng/ml), HMGB1 at 15 and 25 ng/ml or IL-1β+HMGB1, synoviocytes were lysed in 100 μl of buffer (1% Triton X-100, 1% deoxycholic acid, 20 mM NaCl and 25 mM Tris, pH 7.4) and centrifuged at 4°C for 15 minutes at 10,000g .

    Techniques: Activation Assay, Transfection, Construct, Luciferase, Activity Assay

    Effect of HMGB1 and IL-1β on Akt and MAPK phosphorylation . Cells were stimulated with IL-1b (10 ng/ml) for five minutes in the presence or absence of high mobility group box 1 (HMGB1) at 15 and 25 ng/ml. Protein level was determined in cell lysates by western blotting by using specific antibodies against phosphorylated or total proteins. Relative expression of phosphorylated and total protein bands was calculated after densitometric analysis. Data are expressed as mean ± standard error of the mean (samples from three patients). ++ P

    Journal: Arthritis Research & Therapy

    Article Title: High mobility group box 1 potentiates the pro-inflammatory effects of interleukin-1? in osteoarthritic synoviocytes

    doi: 10.1186/ar3124

    Figure Lengend Snippet: Effect of HMGB1 and IL-1β on Akt and MAPK phosphorylation . Cells were stimulated with IL-1b (10 ng/ml) for five minutes in the presence or absence of high mobility group box 1 (HMGB1) at 15 and 25 ng/ml. Protein level was determined in cell lysates by western blotting by using specific antibodies against phosphorylated or total proteins. Relative expression of phosphorylated and total protein bands was calculated after densitometric analysis. Data are expressed as mean ± standard error of the mean (samples from three patients). ++ P

    Article Snippet: Western blot analysis After stimulation for five minutes with IL-1β (10 ng/ml), HMGB1 at 15 and 25 ng/ml or IL-1β+HMGB1, synoviocytes were lysed in 100 μl of buffer (1% Triton X-100, 1% deoxycholic acid, 20 mM NaCl and 25 mM Tris, pH 7.4) and centrifuged at 4°C for 15 minutes at 10,000g .

    Techniques: Western Blot, Expressing

    Confocal analysis of HMGB1 expression in human synovium . Representative immunohistochemical sections of human (a, c) normal ( n = 3) and (b, d) osteoarthritic ( n = 3) synovium with high mobility group box 1 (HMGB1) revealed in fluorescence and read with confocal microscopy. (e) represents immunohistochemistry revealed in 3,3'-diaminobenzidine (DAB) of human osteoarthritic synovium infiltrates, and (f) is as (e) but revealed with the fluorescent antibody. The colour green represents HMGB1 and blue the nucleus. Original magnification × 100. (c) and (d) were amplified by the image processor software.

    Journal: Arthritis Research & Therapy

    Article Title: High mobility group box 1 potentiates the pro-inflammatory effects of interleukin-1? in osteoarthritic synoviocytes

    doi: 10.1186/ar3124

    Figure Lengend Snippet: Confocal analysis of HMGB1 expression in human synovium . Representative immunohistochemical sections of human (a, c) normal ( n = 3) and (b, d) osteoarthritic ( n = 3) synovium with high mobility group box 1 (HMGB1) revealed in fluorescence and read with confocal microscopy. (e) represents immunohistochemistry revealed in 3,3'-diaminobenzidine (DAB) of human osteoarthritic synovium infiltrates, and (f) is as (e) but revealed with the fluorescent antibody. The colour green represents HMGB1 and blue the nucleus. Original magnification × 100. (c) and (d) were amplified by the image processor software.

    Article Snippet: Western blot analysis After stimulation for five minutes with IL-1β (10 ng/ml), HMGB1 at 15 and 25 ng/ml or IL-1β+HMGB1, synoviocytes were lysed in 100 μl of buffer (1% Triton X-100, 1% deoxycholic acid, 20 mM NaCl and 25 mM Tris, pH 7.4) and centrifuged at 4°C for 15 minutes at 10,000g .

    Techniques: Expressing, Immunohistochemistry, Fluorescence, Confocal Microscopy, Amplification, Software

    Effect of HMGB1 and IL-1β on the levels of cytokine and chemokine released into the medium by osteoarthritic synoviocytes . (a) IL-6, (b) IL-8, (c) CCL2 and (d) CCL20 protein levels. Cells were stimulated with IL-1b (10 ng/ml) for 24 hours in the presence or absence of high mobility group box 1 (HMGB1) at 15 and 25 ng/ml. Protein levels were determined in supernatants by ELISA. Data are expressed as mean ± standard error of the mean. Duplicate samples from six patients were used. ++ P

    Journal: Arthritis Research & Therapy

    Article Title: High mobility group box 1 potentiates the pro-inflammatory effects of interleukin-1? in osteoarthritic synoviocytes

    doi: 10.1186/ar3124

    Figure Lengend Snippet: Effect of HMGB1 and IL-1β on the levels of cytokine and chemokine released into the medium by osteoarthritic synoviocytes . (a) IL-6, (b) IL-8, (c) CCL2 and (d) CCL20 protein levels. Cells were stimulated with IL-1b (10 ng/ml) for 24 hours in the presence or absence of high mobility group box 1 (HMGB1) at 15 and 25 ng/ml. Protein levels were determined in supernatants by ELISA. Data are expressed as mean ± standard error of the mean. Duplicate samples from six patients were used. ++ P

    Article Snippet: Western blot analysis After stimulation for five minutes with IL-1β (10 ng/ml), HMGB1 at 15 and 25 ng/ml or IL-1β+HMGB1, synoviocytes were lysed in 100 μl of buffer (1% Triton X-100, 1% deoxycholic acid, 20 mM NaCl and 25 mM Tris, pH 7.4) and centrifuged at 4°C for 15 minutes at 10,000g .

    Techniques: Enzyme-linked Immunosorbent Assay

    Effect of HMGB1 and IL-1β on cytokine and chemokine mRNA levels in OA synoviocytes . (a) IL-6, IL-8, and (b) CCL2 and CCL20 mRNA relative expression. Cells were stimulated with IL-1b (10 ng/ml) for 24 hours in the presence or absence of high mobility group box 1 (HMGB1) at 15 and 25 ng/ml. mRNA expression was determined by real-time PCR. Data are expressed as mean ± standard error of the mean. Duplicate samples from four patients were used. ++ P

    Journal: Arthritis Research & Therapy

    Article Title: High mobility group box 1 potentiates the pro-inflammatory effects of interleukin-1? in osteoarthritic synoviocytes

    doi: 10.1186/ar3124

    Figure Lengend Snippet: Effect of HMGB1 and IL-1β on cytokine and chemokine mRNA levels in OA synoviocytes . (a) IL-6, IL-8, and (b) CCL2 and CCL20 mRNA relative expression. Cells were stimulated with IL-1b (10 ng/ml) for 24 hours in the presence or absence of high mobility group box 1 (HMGB1) at 15 and 25 ng/ml. mRNA expression was determined by real-time PCR. Data are expressed as mean ± standard error of the mean. Duplicate samples from four patients were used. ++ P

    Article Snippet: Western blot analysis After stimulation for five minutes with IL-1β (10 ng/ml), HMGB1 at 15 and 25 ng/ml or IL-1β+HMGB1, synoviocytes were lysed in 100 μl of buffer (1% Triton X-100, 1% deoxycholic acid, 20 mM NaCl and 25 mM Tris, pH 7.4) and centrifuged at 4°C for 15 minutes at 10,000g .

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    Effect of HMGB1 and IL-1β on MMP mRNA levels in osteoarthritic synoviocytes and MMP activity released into the medium . (a) Matrix metalloproteinase (MMP)-1, MMP-3 and MMP-13 mRNA levels and (b) MMP activity. Cells were stimulated with IL-1b (10 ng/ml) for 24 hours in the presence or absence of high mobility group box 1 (HMGB1) at 15 and 25 ng/m. mRNA was determined by real-time PCR and MMP activity was measured in supernatants by a fluorometric procedure, as indicated in Materials and Methods. Data are expressed as mean ± standard error of the mean. Duplicate samples from four patients were used. ++ P

    Journal: Arthritis Research & Therapy

    Article Title: High mobility group box 1 potentiates the pro-inflammatory effects of interleukin-1? in osteoarthritic synoviocytes

    doi: 10.1186/ar3124

    Figure Lengend Snippet: Effect of HMGB1 and IL-1β on MMP mRNA levels in osteoarthritic synoviocytes and MMP activity released into the medium . (a) Matrix metalloproteinase (MMP)-1, MMP-3 and MMP-13 mRNA levels and (b) MMP activity. Cells were stimulated with IL-1b (10 ng/ml) for 24 hours in the presence or absence of high mobility group box 1 (HMGB1) at 15 and 25 ng/m. mRNA was determined by real-time PCR and MMP activity was measured in supernatants by a fluorometric procedure, as indicated in Materials and Methods. Data are expressed as mean ± standard error of the mean. Duplicate samples from four patients were used. ++ P

    Article Snippet: Western blot analysis After stimulation for five minutes with IL-1β (10 ng/ml), HMGB1 at 15 and 25 ng/ml or IL-1β+HMGB1, synoviocytes were lysed in 100 μl of buffer (1% Triton X-100, 1% deoxycholic acid, 20 mM NaCl and 25 mM Tris, pH 7.4) and centrifuged at 4°C for 15 minutes at 10,000g .

    Techniques: Activity Assay, Real-time Polymerase Chain Reaction

    Expression of HMGB1 protein in human synovium . (a) Representative immunohistochemical sections of normal ( n = 3) and osteoarthritic ( n = 3) human synovium with high mobility group box 1 (HMGB1) revealed in 3,3'-diaminobenzidine (DAB). Arrows indicate HMGB1 positive cells. Original magnification × 100. (b) Histograms of normal and osteoarthritic positive cells (%) in the synovial lining cell nucleus and cytoplasm and vicinity. Data are expressed as mean ± standard error of the mean ( n = 3). * P

    Journal: Arthritis Research & Therapy

    Article Title: High mobility group box 1 potentiates the pro-inflammatory effects of interleukin-1? in osteoarthritic synoviocytes

    doi: 10.1186/ar3124

    Figure Lengend Snippet: Expression of HMGB1 protein in human synovium . (a) Representative immunohistochemical sections of normal ( n = 3) and osteoarthritic ( n = 3) human synovium with high mobility group box 1 (HMGB1) revealed in 3,3'-diaminobenzidine (DAB). Arrows indicate HMGB1 positive cells. Original magnification × 100. (b) Histograms of normal and osteoarthritic positive cells (%) in the synovial lining cell nucleus and cytoplasm and vicinity. Data are expressed as mean ± standard error of the mean ( n = 3). * P

    Article Snippet: Western blot analysis After stimulation for five minutes with IL-1β (10 ng/ml), HMGB1 at 15 and 25 ng/ml or IL-1β+HMGB1, synoviocytes were lysed in 100 μl of buffer (1% Triton X-100, 1% deoxycholic acid, 20 mM NaCl and 25 mM Tris, pH 7.4) and centrifuged at 4°C for 15 minutes at 10,000g .

    Techniques: Expressing, Immunohistochemistry

    HMGB-1–induced changes in the expression of SVEC surface adhesion molecules. (A) Representative immunofluorescent staining of β1-integrin in untreated and TNF-α, SDF-1α or HMGB-1–treated SVEC. (B) Number of SVEC showing β1-integrin polarization within the groups. (C) Representative images of P-selectin expression on SVEC plasmalemma in untreated and TNF-α, SDF-1α or HMGB-1–treated cells. (D) Number of SVEC showing P-selectin redistribution after stimulation with different cytokines. (E) Confocal images of ICAM-1 expression patterns on SVEC membrane. (F) Quantity of cells presenting ICAM-1 polarization after preconditioning with TNF-α, SDF-1α or HMGB-1 (* P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: HMGB-1 induces c-kit+ cell microvascular rolling and adhesion via both toll-like receptor-2 and toll-like receptor-4 of endothelial cells

    doi: 10.1111/j.1582-4934.2011.01381.x

    Figure Lengend Snippet: HMGB-1–induced changes in the expression of SVEC surface adhesion molecules. (A) Representative immunofluorescent staining of β1-integrin in untreated and TNF-α, SDF-1α or HMGB-1–treated SVEC. (B) Number of SVEC showing β1-integrin polarization within the groups. (C) Representative images of P-selectin expression on SVEC plasmalemma in untreated and TNF-α, SDF-1α or HMGB-1–treated cells. (D) Number of SVEC showing P-selectin redistribution after stimulation with different cytokines. (E) Confocal images of ICAM-1 expression patterns on SVEC membrane. (F) Quantity of cells presenting ICAM-1 polarization after preconditioning with TNF-α, SDF-1α or HMGB-1 (* P

    Article Snippet: It is reasonable to speculate HMGB-1 mediates c-kit+ cell recruitment via both the TLR-2 and TLR-4 signalling of endothelial cells.

    Techniques: Expressing, Staining

    The mRNA level of P-selectin in presence or absence of TLR-2 and TLR-4. Quantitative real-time PCR analysis in ‘Control’, ‘HMGB-1’, ‘TLR-2ko’ and ‘TLR-4ko’ groups. P-selectin mRNA is significantly up-regulated by HMGB-1 only when TLR-2 is functional. The average mRNA expression level of eNOS and c-kit in ‘Control’ cremasters was arbitrarily given a value of 1 (line) (** P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: HMGB-1 induces c-kit+ cell microvascular rolling and adhesion via both toll-like receptor-2 and toll-like receptor-4 of endothelial cells

    doi: 10.1111/j.1582-4934.2011.01381.x

    Figure Lengend Snippet: The mRNA level of P-selectin in presence or absence of TLR-2 and TLR-4. Quantitative real-time PCR analysis in ‘Control’, ‘HMGB-1’, ‘TLR-2ko’ and ‘TLR-4ko’ groups. P-selectin mRNA is significantly up-regulated by HMGB-1 only when TLR-2 is functional. The average mRNA expression level of eNOS and c-kit in ‘Control’ cremasters was arbitrarily given a value of 1 (line) (** P

    Article Snippet: It is reasonable to speculate HMGB-1 mediates c-kit+ cell recruitment via both the TLR-2 and TLR-4 signalling of endothelial cells.

    Techniques: Real-time Polymerase Chain Reaction, Functional Assay, Expressing

    HMGB-1–mediated c-kit + cell rolling and adhesion on cremaster muscle. (A) Percentage of rolling WT c-kit + cells in TLRs knockout mice. (B) Quantity of adherent WT c-kit + cells in TLRs knockout mice. (C) Percentage of rolling TLR-2 (−/−) and Tlr4 (LPS-del)c-kit + cells in WT animals. (D) Number of adherent TLR-2 (−/−) and Tlr4 (LPS-del) c-kit + cells in WT animals (* P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: HMGB-1 induces c-kit+ cell microvascular rolling and adhesion via both toll-like receptor-2 and toll-like receptor-4 of endothelial cells

    doi: 10.1111/j.1582-4934.2011.01381.x

    Figure Lengend Snippet: HMGB-1–mediated c-kit + cell rolling and adhesion on cremaster muscle. (A) Percentage of rolling WT c-kit + cells in TLRs knockout mice. (B) Quantity of adherent WT c-kit + cells in TLRs knockout mice. (C) Percentage of rolling TLR-2 (−/−) and Tlr4 (LPS-del)c-kit + cells in WT animals. (D) Number of adherent TLR-2 (−/−) and Tlr4 (LPS-del) c-kit + cells in WT animals (* P

    Article Snippet: It is reasonable to speculate HMGB-1 mediates c-kit+ cell recruitment via both the TLR-2 and TLR-4 signalling of endothelial cells.

    Techniques: Knock-Out, Mouse Assay

    HMGB-1 increases eNOS and c-kit expression in presence of TLR-2. Quantitative real-time PCR and confocal microscopy analysis of stem cell homing signals in cremaster muscle. (A) eNOS and c-kit genes expression in ‘Control’, ‘HMGB-1’, ‘TLR-2ko’ and ‘TLR-4ko’ groups. The average mRNA expression level of eNOS and c-kit in ‘Control’ cremaster muscle tissue was arbitrarily given a value of 1 (line) (* P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: HMGB-1 induces c-kit+ cell microvascular rolling and adhesion via both toll-like receptor-2 and toll-like receptor-4 of endothelial cells

    doi: 10.1111/j.1582-4934.2011.01381.x

    Figure Lengend Snippet: HMGB-1 increases eNOS and c-kit expression in presence of TLR-2. Quantitative real-time PCR and confocal microscopy analysis of stem cell homing signals in cremaster muscle. (A) eNOS and c-kit genes expression in ‘Control’, ‘HMGB-1’, ‘TLR-2ko’ and ‘TLR-4ko’ groups. The average mRNA expression level of eNOS and c-kit in ‘Control’ cremaster muscle tissue was arbitrarily given a value of 1 (line) (* P

    Article Snippet: It is reasonable to speculate HMGB-1 mediates c-kit+ cell recruitment via both the TLR-2 and TLR-4 signalling of endothelial cells.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Confocal Microscopy

    Endogenous leukocyte adhesion on cremaster muscle 15 min. after direct HMGB-1 superfusion. (A) Number (n/mm 2 ) of adherent leukocytes in ‘Control’ and ‘HMGB-1’ groups. (B) Quantitative real-time PCR analysis of the pro-inflammatory signals β1-integrin and CD45 of ‘Control’, ‘HMGB-1’, ‘TLR-2ko’ and ‘TLR-4ko’ groups. The average mRNA expression level of β1-intergrin and CD45 and c-kit in ‘Control’ cremasters was arbitrarily given a value of 1 (line) (* P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: HMGB-1 induces c-kit+ cell microvascular rolling and adhesion via both toll-like receptor-2 and toll-like receptor-4 of endothelial cells

    doi: 10.1111/j.1582-4934.2011.01381.x

    Figure Lengend Snippet: Endogenous leukocyte adhesion on cremaster muscle 15 min. after direct HMGB-1 superfusion. (A) Number (n/mm 2 ) of adherent leukocytes in ‘Control’ and ‘HMGB-1’ groups. (B) Quantitative real-time PCR analysis of the pro-inflammatory signals β1-integrin and CD45 of ‘Control’, ‘HMGB-1’, ‘TLR-2ko’ and ‘TLR-4ko’ groups. The average mRNA expression level of β1-intergrin and CD45 and c-kit in ‘Control’ cremasters was arbitrarily given a value of 1 (line) (* P

    Article Snippet: It is reasonable to speculate HMGB-1 mediates c-kit+ cell recruitment via both the TLR-2 and TLR-4 signalling of endothelial cells.

    Techniques: Real-time Polymerase Chain Reaction, Expressing

    Effects of direct HMGB-1 superfusion of cremaster muscle on c-kit + cell rolling and adhesion 15 min. after treatment. (A) Representative intravital fluorescence microscopy images of rolling and adherent c-kit + cells. (B) Percentage of rolling c-kit + cells in ‘Control’, ‘HMGB-1’, ‘LPS’ and ‘MALP-2’ groups. (C) Amount of adherent c-kit + cells in ‘Control’, ‘HMGB-1’, ‘LPS’ and ‘MALP-2’ groups (* P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: HMGB-1 induces c-kit+ cell microvascular rolling and adhesion via both toll-like receptor-2 and toll-like receptor-4 of endothelial cells

    doi: 10.1111/j.1582-4934.2011.01381.x

    Figure Lengend Snippet: Effects of direct HMGB-1 superfusion of cremaster muscle on c-kit + cell rolling and adhesion 15 min. after treatment. (A) Representative intravital fluorescence microscopy images of rolling and adherent c-kit + cells. (B) Percentage of rolling c-kit + cells in ‘Control’, ‘HMGB-1’, ‘LPS’ and ‘MALP-2’ groups. (C) Amount of adherent c-kit + cells in ‘Control’, ‘HMGB-1’, ‘LPS’ and ‘MALP-2’ groups (* P

    Article Snippet: It is reasonable to speculate HMGB-1 mediates c-kit+ cell recruitment via both the TLR-2 and TLR-4 signalling of endothelial cells.

    Techniques: Fluorescence, Microscopy

    Effects of siRNA on 8-nitroG formation in indium-treated A549 cells. ( A ) Reduction in HMGB1, RAGE and TLR9 expression by siRNA transfection into A549 cells. Effects of siRNA on protein expression were evaluated by Western blotting. These blots were cropped from different parts in the same gel, and each blot was divided with white lines. Full-length blots are shown in Supplementary Figure S2 online. ( B ) Image analysis for HMGB1, RAGE and TLR9 expression in siRNA-transfected A549 cells. These values were expressed as fold changes compared with control. ( C ) Fluorescent images of 8-nitroG formation in indium-treated A549 cells and effects of siRNA. Cells were transfected with 10 nM siRNA for HMGB1 , AGER and TLR9 or negative control siRNA for 2 days and then treated with 200 ng/ml indium compounds for 4 h as described in “ Methods ” section. 8-NitroG formation was evaluated by immunocytochemistry as described in “ Methods ” section. The nucleus was stained with Hoechst 33258. Magnification, × 200. ( D ) Quantitative image analysis for the effects of siRNA on 8-nitroG formation in indium-treated A549 cells. Staining intensities of 8-nitroG per area were analyzed with an image J software. The relative intensity of the control was set at 1. ( B , D ) The data were expressed as means ± SD of 3–4 independent experiments. ** p

    Journal: Scientific Reports

    Article Title: Nitrative DNA damage in lung epithelial cells exposed to indium nanoparticles and indium ions

    doi: 10.1038/s41598-020-67488-3

    Figure Lengend Snippet: Effects of siRNA on 8-nitroG formation in indium-treated A549 cells. ( A ) Reduction in HMGB1, RAGE and TLR9 expression by siRNA transfection into A549 cells. Effects of siRNA on protein expression were evaluated by Western blotting. These blots were cropped from different parts in the same gel, and each blot was divided with white lines. Full-length blots are shown in Supplementary Figure S2 online. ( B ) Image analysis for HMGB1, RAGE and TLR9 expression in siRNA-transfected A549 cells. These values were expressed as fold changes compared with control. ( C ) Fluorescent images of 8-nitroG formation in indium-treated A549 cells and effects of siRNA. Cells were transfected with 10 nM siRNA for HMGB1 , AGER and TLR9 or negative control siRNA for 2 days and then treated with 200 ng/ml indium compounds for 4 h as described in “ Methods ” section. 8-NitroG formation was evaluated by immunocytochemistry as described in “ Methods ” section. The nucleus was stained with Hoechst 33258. Magnification, × 200. ( D ) Quantitative image analysis for the effects of siRNA on 8-nitroG formation in indium-treated A549 cells. Staining intensities of 8-nitroG per area were analyzed with an image J software. The relative intensity of the control was set at 1. ( B , D ) The data were expressed as means ± SD of 3–4 independent experiments. ** p

    Article Snippet: Then the membrane was incubated with anti-HMGB1 mouse monoclonal antibody (1:500, ab77302, Abcam, Cambridge, UK), anti-RAGE mouse monoclonal antibody (1:500, ab54741, Abcam) or anti-TLR9 rabbit polyclonal antibody (1:500, ab37154, Abcam) along with anti-GAPDH rabbit polyclonal antibody (1:1,000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h. The membrane was then treated with horseradish peroxidase-conjugated anti-rabbit IgG antibody (1:10,000; Santa Cruz Biotechnology) and/or anti-mouse IgG antibody (1:2,000, Santa Cruz Biotechnology) for 30 min.

    Techniques: Expressing, Transfection, Western Blot, Negative Control, Immunocytochemistry, Staining, Software

    Effects of HMGB1 and RAGE antibodies on 8-nitroG formation in indium-treated A549 cells. ( A ) Fluorescent images of 8-nitroG formation in indium-treated A549 cells and effects of antibodies. A549 cells were pretreated with 10 µg/ml of anti-HMGB1 and anti-RAGE antbodies and their isotype control IgGs for 30 min, followed by the treatment with 200 ng/ml of In 2 O 3 , ITO and InCl 3 as described in “ Methods ” section. 8-NitroG was detected by immunocytochemistry. The nucleus was stained with Hoechst 33258. Magnification × 200. ( B ) Quantitative image analysis for the effects of antbodies on 8-nitroG formation in indium-treated A549 cells. Staining intensities of 8-nitroG per area were analyzed with an ImageJ software. The relative intensity of the control was set at 1. The data were expressed as means ± SD of 3–4 independent experiments. ** p

    Journal: Scientific Reports

    Article Title: Nitrative DNA damage in lung epithelial cells exposed to indium nanoparticles and indium ions

    doi: 10.1038/s41598-020-67488-3

    Figure Lengend Snippet: Effects of HMGB1 and RAGE antibodies on 8-nitroG formation in indium-treated A549 cells. ( A ) Fluorescent images of 8-nitroG formation in indium-treated A549 cells and effects of antibodies. A549 cells were pretreated with 10 µg/ml of anti-HMGB1 and anti-RAGE antbodies and their isotype control IgGs for 30 min, followed by the treatment with 200 ng/ml of In 2 O 3 , ITO and InCl 3 as described in “ Methods ” section. 8-NitroG was detected by immunocytochemistry. The nucleus was stained with Hoechst 33258. Magnification × 200. ( B ) Quantitative image analysis for the effects of antbodies on 8-nitroG formation in indium-treated A549 cells. Staining intensities of 8-nitroG per area were analyzed with an ImageJ software. The relative intensity of the control was set at 1. The data were expressed as means ± SD of 3–4 independent experiments. ** p

    Article Snippet: Then the membrane was incubated with anti-HMGB1 mouse monoclonal antibody (1:500, ab77302, Abcam, Cambridge, UK), anti-RAGE mouse monoclonal antibody (1:500, ab54741, Abcam) or anti-TLR9 rabbit polyclonal antibody (1:500, ab37154, Abcam) along with anti-GAPDH rabbit polyclonal antibody (1:1,000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h. The membrane was then treated with horseradish peroxidase-conjugated anti-rabbit IgG antibody (1:10,000; Santa Cruz Biotechnology) and/or anti-mouse IgG antibody (1:2,000, Santa Cruz Biotechnology) for 30 min.

    Techniques: Immunocytochemistry, Staining, Software

    Conditional knockout of HMGB1 in rods does not affect microglial activation or mobilization. ( A ) Retinas of HMGB1ΔRod and control mice were sectioned at a 30-µm thickness and probed with anti–Iba-1 antibody to view activation and mobilization of microglia/macrophage at 7 dprd. Nuclei were counterstained with DAPI. Scale bar : 50 µm. ( B ) Entire retinas of HMGB1ΔRod and control mice were probed with anti–Iba-1 antibody and flat-mounted for confocal imaging at 7 dprd. Multiple confocal images were Z-stacked to create merged images of different layers of the retina based on the DAPI staining. Scale bar : 200 µm. ( C ) Supernatants from eyecups were collected and the levels of HMGB1 in HMGB1ΔRod and control mice at 3 and 7 dprd were measured by ELISA. One-way ANOVA ( n = 4). IPL, inner plexiform layer; IS/OS, inner and outer segment; ns, not significant; OPL, outer plexiform layer.

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Loss of High-Mobility Group Box 1 (HMGB1) Protein in Rods Accelerates Rod Photoreceptor Degeneration After Retinal Detachment

    doi: 10.1167/iovs.61.5.50

    Figure Lengend Snippet: Conditional knockout of HMGB1 in rods does not affect microglial activation or mobilization. ( A ) Retinas of HMGB1ΔRod and control mice were sectioned at a 30-µm thickness and probed with anti–Iba-1 antibody to view activation and mobilization of microglia/macrophage at 7 dprd. Nuclei were counterstained with DAPI. Scale bar : 50 µm. ( B ) Entire retinas of HMGB1ΔRod and control mice were probed with anti–Iba-1 antibody and flat-mounted for confocal imaging at 7 dprd. Multiple confocal images were Z-stacked to create merged images of different layers of the retina based on the DAPI staining. Scale bar : 200 µm. ( C ) Supernatants from eyecups were collected and the levels of HMGB1 in HMGB1ΔRod and control mice at 3 and 7 dprd were measured by ELISA. One-way ANOVA ( n = 4). IPL, inner plexiform layer; IS/OS, inner and outer segment; ns, not significant; OPL, outer plexiform layer.

    Article Snippet: Notably, the Cre IF staining in PRs colocalized with HMGB1 and replicated the ring-like nuclear pattern in these cells.

    Techniques: Knock-Out, Activation Assay, Mouse Assay, Imaging, Staining, Enzyme-linked Immunosorbent Assay

    Conditional knockout of HMGB1 from rods accelerates their degeneration during RD. ( A ) OCT images in attached and detached retinas at 4 and 8 weeks post-RD in HMGB1ΔRod and control mice. Due to the angle created by retinal detachment, a novel method was developed to measure the thickness of the retina to ensure that corresponding points on the retinas were measured (see Materials and Methods). ( B ) The thickness of ONL and the thickness from the ILM to the OLM were measured at 500 pixels away from the optic nerve head using ImageJ. The ONL/(ILM-OLM) ratio was calculated and graphed. ( C ) Hematoxylin and eosin staining of retinal sections was performed at 10 weeks post-RD in HMGB1ΔRod and control mice. ( D ) The nuclei count in the ONL in the detached portion of retinal sections was performed using ImageJ. The ONL area and the area between the ILM and OLM in the detached portion of the retina were also measured. The ONL/(ILM-OLM) area ratio was calculated. Normalized data were graphed with controls set as 100%. ( E ) Retinal sections of HMGB1ΔRod and control mice at 10 weeks post-RD were probed with antirhodopsin Ab. Nuclei were counterstained with DAPI. Scale bars : 50 µm. ** P

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Loss of High-Mobility Group Box 1 (HMGB1) Protein in Rods Accelerates Rod Photoreceptor Degeneration After Retinal Detachment

    doi: 10.1167/iovs.61.5.50

    Figure Lengend Snippet: Conditional knockout of HMGB1 from rods accelerates their degeneration during RD. ( A ) OCT images in attached and detached retinas at 4 and 8 weeks post-RD in HMGB1ΔRod and control mice. Due to the angle created by retinal detachment, a novel method was developed to measure the thickness of the retina to ensure that corresponding points on the retinas were measured (see Materials and Methods). ( B ) The thickness of ONL and the thickness from the ILM to the OLM were measured at 500 pixels away from the optic nerve head using ImageJ. The ONL/(ILM-OLM) ratio was calculated and graphed. ( C ) Hematoxylin and eosin staining of retinal sections was performed at 10 weeks post-RD in HMGB1ΔRod and control mice. ( D ) The nuclei count in the ONL in the detached portion of retinal sections was performed using ImageJ. The ONL area and the area between the ILM and OLM in the detached portion of the retina were also measured. The ONL/(ILM-OLM) area ratio was calculated. Normalized data were graphed with controls set as 100%. ( E ) Retinal sections of HMGB1ΔRod and control mice at 10 weeks post-RD were probed with antirhodopsin Ab. Nuclei were counterstained with DAPI. Scale bars : 50 µm. ** P

    Article Snippet: Notably, the Cre IF staining in PRs colocalized with HMGB1 and replicated the ring-like nuclear pattern in these cells.

    Techniques: Knock-Out, Mouse Assay, Staining

    Conditional knockout of HMGB1 in rod photoreceptors does not affect retinal thickness or ERG response at baseline. ( A ) Fellow and detached retinas at 7 dprd were probed with HMGB1 Ab to confirm the complete and specific knockout of HMGB1 in rod photoreceptors in HMGB1ΔRod mice. Rho-Cre + mice served as controls. Nuclei were counterstained with DAPI. Arrowheads show the infiltrated cells positive for HMGB1 IF. Scale bar : 50 µm. ( B – D ) OCT imaging and ERG analyses were performed in naive HMGB1ΔRod and control mice at 20 weeks of age. Represented OCT images are shown in ( B ). The thicknesses of the total retina and ONL were measured at four points (nasal, temporal, superior, and inferior) at a distance of 500 µm from the optic nerve head. Represented ERG response traces are shown in ( C ). The amplitudes of scotopic a-wave and b-wave and photopic b-wave at the intensity of 32 cd•s/m 2 are shown in ( D ). Control mice, n = 11; HMGB1ΔRod mice, n = 6. Data were plotted with mean ± SD. Paired Student's t -test. ns, not significant.

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Loss of High-Mobility Group Box 1 (HMGB1) Protein in Rods Accelerates Rod Photoreceptor Degeneration After Retinal Detachment

    doi: 10.1167/iovs.61.5.50

    Figure Lengend Snippet: Conditional knockout of HMGB1 in rod photoreceptors does not affect retinal thickness or ERG response at baseline. ( A ) Fellow and detached retinas at 7 dprd were probed with HMGB1 Ab to confirm the complete and specific knockout of HMGB1 in rod photoreceptors in HMGB1ΔRod mice. Rho-Cre + mice served as controls. Nuclei were counterstained with DAPI. Arrowheads show the infiltrated cells positive for HMGB1 IF. Scale bar : 50 µm. ( B – D ) OCT imaging and ERG analyses were performed in naive HMGB1ΔRod and control mice at 20 weeks of age. Represented OCT images are shown in ( B ). The thicknesses of the total retina and ONL were measured at four points (nasal, temporal, superior, and inferior) at a distance of 500 µm from the optic nerve head. Represented ERG response traces are shown in ( C ). The amplitudes of scotopic a-wave and b-wave and photopic b-wave at the intensity of 32 cd•s/m 2 are shown in ( D ). Control mice, n = 11; HMGB1ΔRod mice, n = 6. Data were plotted with mean ± SD. Paired Student's t -test. ns, not significant.

    Article Snippet: Notably, the Cre IF staining in PRs colocalized with HMGB1 and replicated the ring-like nuclear pattern in these cells.

    Techniques: Knock-Out, Mouse Assay, Imaging

    The expression pattern of HMGB1 in the mouse retina. ( A ) Naive retinas of 8-week-old C57BL/6J mice were probed with anti-HMGB1 antibody (Ab). Note low HMGB1 Ab IF in most of the nuclei in the ONL. ( B ) Naive retinas of C57BL/6 mice were probed with anti-HMGB1 Ab ( green ) and PNA ( red ). Note juxtaposition of PNA binding (indicating cone inner and outer segments) with a minority of photoreceptor nuclei with high HMGB1 Ab binding. ( C ) Colocalization of HMGB1 ( green ) and Cre ( red ) in the naive retinas of opsin-Cre and rhodopsin-Cre (Rho-cre) mice on a C57BL/6J background. Note the colocalization of high HMGB1 IF with opsin-Cre IF but no colocalization with rho-Cre IF. ( D ) Naive retinas of Nrl −/− mice (cone-like photoreceptors only) were probed with anti-HMGB1 Ab and nuclei were counterstained with DAPI. Note that HMGB1 IF is comparable in the ONL and INL. GCL, ganglion cell layer; INL, inner nuclear layer; IS/OS, inner and outer segments. Scale bars : 50 µm.

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Loss of High-Mobility Group Box 1 (HMGB1) Protein in Rods Accelerates Rod Photoreceptor Degeneration After Retinal Detachment

    doi: 10.1167/iovs.61.5.50

    Figure Lengend Snippet: The expression pattern of HMGB1 in the mouse retina. ( A ) Naive retinas of 8-week-old C57BL/6J mice were probed with anti-HMGB1 antibody (Ab). Note low HMGB1 Ab IF in most of the nuclei in the ONL. ( B ) Naive retinas of C57BL/6 mice were probed with anti-HMGB1 Ab ( green ) and PNA ( red ). Note juxtaposition of PNA binding (indicating cone inner and outer segments) with a minority of photoreceptor nuclei with high HMGB1 Ab binding. ( C ) Colocalization of HMGB1 ( green ) and Cre ( red ) in the naive retinas of opsin-Cre and rhodopsin-Cre (Rho-cre) mice on a C57BL/6J background. Note the colocalization of high HMGB1 IF with opsin-Cre IF but no colocalization with rho-Cre IF. ( D ) Naive retinas of Nrl −/− mice (cone-like photoreceptors only) were probed with anti-HMGB1 Ab and nuclei were counterstained with DAPI. Note that HMGB1 IF is comparable in the ONL and INL. GCL, ganglion cell layer; INL, inner nuclear layer; IS/OS, inner and outer segments. Scale bars : 50 µm.

    Article Snippet: Notably, the Cre IF staining in PRs colocalized with HMGB1 and replicated the ring-like nuclear pattern in these cells.

    Techniques: Expressing, Mouse Assay, Binding Assay

    RD triggers the translocation and release of HMGB1. RD was created by subretinal injection of 1% hyaluronic acid in the left eyes of C57BL/6 mice. The contralateral eyes did not receive any treatment and served as fellow controls. ( A ) Costaining of HMGB1 Ab ( green ) and PNA ( red ) in the naive retina and detached retinas at 3 and 7 dprd. Note the relatively low HMGB1 IF in most PR nuclei and the spatial juxtaposition of high HMGB1 IF in some PR nuclei with PNA staining of cone inner and outer segments. Scale bar : 50 µm. ( B – D ) Mouse eyes were enucleated at 1, 2, 3, and 7 dprd. Eyecups were minced and supernatants collected for ELISA to measure the levels of soluble HMGB1. ( B ) Insoluble eyecup pieces underwent extraction of nuclear and cytoplasmic proteins for Western blot. Lamin A/C and glyceraldehyde 3-phosphate dehydrogenase were used as loading controls for the nuclear fraction and cytoplasmic fraction, respectively. Blots on the right side show the relative amounts of HMGB1 protein in cytoplasmic and nuclear fractions in naive retinas and the efficiency of fractionization. ( C ) Graphs show the quantification of protein levels based on densitometry of the Western blots ( n = 3). ( D ) Graphs show levels of HMGB1 in the eyecup supernatant ( n = 4) measured by ELISA. Data were plotted with mean ± SD. One-way ANOVA. *Indicates significant difference in comparison with naive. Statistical significance was set at P

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Loss of High-Mobility Group Box 1 (HMGB1) Protein in Rods Accelerates Rod Photoreceptor Degeneration After Retinal Detachment

    doi: 10.1167/iovs.61.5.50

    Figure Lengend Snippet: RD triggers the translocation and release of HMGB1. RD was created by subretinal injection of 1% hyaluronic acid in the left eyes of C57BL/6 mice. The contralateral eyes did not receive any treatment and served as fellow controls. ( A ) Costaining of HMGB1 Ab ( green ) and PNA ( red ) in the naive retina and detached retinas at 3 and 7 dprd. Note the relatively low HMGB1 IF in most PR nuclei and the spatial juxtaposition of high HMGB1 IF in some PR nuclei with PNA staining of cone inner and outer segments. Scale bar : 50 µm. ( B – D ) Mouse eyes were enucleated at 1, 2, 3, and 7 dprd. Eyecups were minced and supernatants collected for ELISA to measure the levels of soluble HMGB1. ( B ) Insoluble eyecup pieces underwent extraction of nuclear and cytoplasmic proteins for Western blot. Lamin A/C and glyceraldehyde 3-phosphate dehydrogenase were used as loading controls for the nuclear fraction and cytoplasmic fraction, respectively. Blots on the right side show the relative amounts of HMGB1 protein in cytoplasmic and nuclear fractions in naive retinas and the efficiency of fractionization. ( C ) Graphs show the quantification of protein levels based on densitometry of the Western blots ( n = 3). ( D ) Graphs show levels of HMGB1 in the eyecup supernatant ( n = 4) measured by ELISA. Data were plotted with mean ± SD. One-way ANOVA. *Indicates significant difference in comparison with naive. Statistical significance was set at P

    Article Snippet: Notably, the Cre IF staining in PRs colocalized with HMGB1 and replicated the ring-like nuclear pattern in these cells.

    Techniques: Translocation Assay, Injection, Mouse Assay, Staining, Enzyme-linked Immunosorbent Assay, Western Blot

    (a) The expression of HMGB1, TNF- α , and IL-6 on cDNA level was detected by real-time PCR ( * P

    Journal: Mediators of Inflammation

    Article Title: Ethyl Pyruvate Ameliorates Hepatic Ischemia-Reperfusion Injury by Inhibiting Intrinsic Pathway of Apoptosis and Autophagy

    doi: 10.1155/2013/461536

    Figure Lengend Snippet: (a) The expression of HMGB1, TNF- α , and IL-6 on cDNA level was detected by real-time PCR ( * P

    Article Snippet: The cell membranes were ruptured with 0.2% Triton X-100 at room temperature for 20 min. Nonspecific antigen binding sites were blocked with 5% BSA and the sections were then incubated overnight with HMGB1 (1 : 1000) at 4°C.

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    In the condition of hepatic ischemia-reperfusion injury, HMGB1 is predominantly released by stressed hepatocytes, which actively secrete HMGB1 into the circulation. After release, HMGB1, as a proinflammatory cytokine, combined with TLR4 was expressed on the surface of hepatocytes or other nonparenchymal cells. NF- κ B signal pathway is further activated to promote cytokine release, such as TNF- α and IL-6. Under the stimulation of TNF- α and IL-6, intrinsic pathway of apoptosis is initiated, exhibited as the upregulation of Bax and downregulation of Bcl-2. On the other hand, with the translocation of HMGB1 into cytoplasm from nuclei, HMGB1 competitively combined with Beclin-1 to promote the level of autophagy through representing the site of Bcl-2, which can maintain the inactive status of autophagy. Ethyl pyruvate successfully inhibits the expression and translocation of HMGB1 in stressed cells under the condition of hepatic I/R injury and further downregulates intrinsic pathway of apoptosis and autophagy to ameliorate hepatic I/R injury.

    Journal: Mediators of Inflammation

    Article Title: Ethyl Pyruvate Ameliorates Hepatic Ischemia-Reperfusion Injury by Inhibiting Intrinsic Pathway of Apoptosis and Autophagy

    doi: 10.1155/2013/461536

    Figure Lengend Snippet: In the condition of hepatic ischemia-reperfusion injury, HMGB1 is predominantly released by stressed hepatocytes, which actively secrete HMGB1 into the circulation. After release, HMGB1, as a proinflammatory cytokine, combined with TLR4 was expressed on the surface of hepatocytes or other nonparenchymal cells. NF- κ B signal pathway is further activated to promote cytokine release, such as TNF- α and IL-6. Under the stimulation of TNF- α and IL-6, intrinsic pathway of apoptosis is initiated, exhibited as the upregulation of Bax and downregulation of Bcl-2. On the other hand, with the translocation of HMGB1 into cytoplasm from nuclei, HMGB1 competitively combined with Beclin-1 to promote the level of autophagy through representing the site of Bcl-2, which can maintain the inactive status of autophagy. Ethyl pyruvate successfully inhibits the expression and translocation of HMGB1 in stressed cells under the condition of hepatic I/R injury and further downregulates intrinsic pathway of apoptosis and autophagy to ameliorate hepatic I/R injury.

    Article Snippet: The cell membranes were ruptured with 0.2% Triton X-100 at room temperature for 20 min. Nonspecific antigen binding sites were blocked with 5% BSA and the sections were then incubated overnight with HMGB1 (1 : 1000) at 4°C.

    Techniques: Translocation Assay, Expressing

    Blood HMGB1 levels increase before epilepsy onset in electrical SE–exposed rats and predict therapeutic response to antiinflammatory drugs. Longitudinal analysis of total HMGB1 and levels of acetylated, reduced, and disulfide isoforms in blood plasma at representative time points of disease development in SE-exposed rats receiving treatment (drug) or corresponding vehicle (see key). Treatment included anakinra+BoxA+ifenprodil (SE+drug) (detailed protocol in Methods; Supplemental Figure 5A ). Data are shown as mean ± SEM ( n = 9 rats each group; dot plots are shown in Supplemental Figure 7 ). Rats are the same as reported in Figure 3 . * P

    Journal: The Journal of Clinical Investigation

    Article Title: Molecular isoforms of high-mobility group box 1 are mechanistic biomarkers for epilepsy

    doi: 10.1172/JCI92001

    Figure Lengend Snippet: Blood HMGB1 levels increase before epilepsy onset in electrical SE–exposed rats and predict therapeutic response to antiinflammatory drugs. Longitudinal analysis of total HMGB1 and levels of acetylated, reduced, and disulfide isoforms in blood plasma at representative time points of disease development in SE-exposed rats receiving treatment (drug) or corresponding vehicle (see key). Treatment included anakinra+BoxA+ifenprodil (SE+drug) (detailed protocol in Methods; Supplemental Figure 5A ). Data are shown as mean ± SEM ( n = 9 rats each group; dot plots are shown in Supplemental Figure 7 ). Rats are the same as reported in Figure 3 . * P

    Article Snippet: Supernatant HMGB1 was immunoprecipitated with 5 μg rabbit anti-HMGB1 (ab18256; Abcam) for 16 hours at 4°C as previously described ( ).

    Techniques:

    HMGB1 levels in blood of drug-resistant and drug-sensitive epilepsy patients and healthy controls. ( A ) Total HMGB1 levels in healthy controls (1.1 ± 0.07 ng/ml, n = 74) and patients with well-controlled epilepsy (1.2 ± 0.15 ng/ml, n = 20) or drug-resistant epilepsy (8.7 ± 0.47 ng/ml, n = 65; P

    Journal: The Journal of Clinical Investigation

    Article Title: Molecular isoforms of high-mobility group box 1 are mechanistic biomarkers for epilepsy

    doi: 10.1172/JCI92001

    Figure Lengend Snippet: HMGB1 levels in blood of drug-resistant and drug-sensitive epilepsy patients and healthy controls. ( A ) Total HMGB1 levels in healthy controls (1.1 ± 0.07 ng/ml, n = 74) and patients with well-controlled epilepsy (1.2 ± 0.15 ng/ml, n = 20) or drug-resistant epilepsy (8.7 ± 0.47 ng/ml, n = 65; P

    Article Snippet: Supernatant HMGB1 was immunoprecipitated with 5 μg rabbit anti-HMGB1 (ab18256; Abcam) for 16 hours at 4°C as previously described ( ).

    Techniques:

    Brain and blood HMGB1 measurements during epileptogenesis evoked by electrical SE in adult rats. ( A ) Representative photomicrographs of hippocampi from control rats (sham) or rats at 3 hours, 6 hours, and 4 days after SE ( n = 5 each group). Top row shows HMGB1 immunoreactivity in cell nuclei (arrows) or in cytoplasm of glial cells (arrowheads). Immunofluorescence panels show localization of HMGB1 signal (green) in OX-42–positive microglia (red), GFAP-positive astrocytes (red), and EBA-positive endothelial cells (red); colocalization signal is depicted in yellow (merge). White arrows depict cytoplasmic staining. Hoechst-positive nuclei are shown in blue. Rad, stratum radiatum; LMol, stratum lacunosum moleculare. Scale bars: 25 μm (top row); 20 μm (bottom row; immunofluorescence panels). ( B ) Levels of HMGB1 isoforms in brain tissue (hippocampus) and corresponding blood of rats during epileptogenesis. Data are shown as mean ± SEM ( n = 5 each group). Dot plots are shown in Supplemental Figure 4 . * P

    Journal: The Journal of Clinical Investigation

    Article Title: Molecular isoforms of high-mobility group box 1 are mechanistic biomarkers for epilepsy

    doi: 10.1172/JCI92001

    Figure Lengend Snippet: Brain and blood HMGB1 measurements during epileptogenesis evoked by electrical SE in adult rats. ( A ) Representative photomicrographs of hippocampi from control rats (sham) or rats at 3 hours, 6 hours, and 4 days after SE ( n = 5 each group). Top row shows HMGB1 immunoreactivity in cell nuclei (arrows) or in cytoplasm of glial cells (arrowheads). Immunofluorescence panels show localization of HMGB1 signal (green) in OX-42–positive microglia (red), GFAP-positive astrocytes (red), and EBA-positive endothelial cells (red); colocalization signal is depicted in yellow (merge). White arrows depict cytoplasmic staining. Hoechst-positive nuclei are shown in blue. Rad, stratum radiatum; LMol, stratum lacunosum moleculare. Scale bars: 25 μm (top row); 20 μm (bottom row; immunofluorescence panels). ( B ) Levels of HMGB1 isoforms in brain tissue (hippocampus) and corresponding blood of rats during epileptogenesis. Data are shown as mean ± SEM ( n = 5 each group). Dot plots are shown in Supplemental Figure 4 . * P

    Article Snippet: Supernatant HMGB1 was immunoprecipitated with 5 μg rabbit anti-HMGB1 (ab18256; Abcam) for 16 hours at 4°C as previously described ( ).

    Techniques: Immunofluorescence, Staining

    Early prediction of epilepsy development by monitoring blood HMGB1 level in lithium+pilocarpine SE rats. ( A ) Longitudinal analysis of total HMGB1 and acetylated, reduced, and disulfide isoform levels in blood at representative time points of disease development (see key in A ). Blood was drawn at 23 days (epileptogenic phase prodromal to epilepsy onset), 73 days (encompassing the time of disease onset in 70% of rats), and 7.5 months (chronic epilepsy). Data represent mean ± SEM; n = 7 sham; n = 5 epileptic (Epi); n = 5 nonepileptic (Nonepi) rats. Dot plots are shown in Supplemental Figure 10 . ** P

    Journal: The Journal of Clinical Investigation

    Article Title: Molecular isoforms of high-mobility group box 1 are mechanistic biomarkers for epilepsy

    doi: 10.1172/JCI92001

    Figure Lengend Snippet: Early prediction of epilepsy development by monitoring blood HMGB1 level in lithium+pilocarpine SE rats. ( A ) Longitudinal analysis of total HMGB1 and acetylated, reduced, and disulfide isoform levels in blood at representative time points of disease development (see key in A ). Blood was drawn at 23 days (epileptogenic phase prodromal to epilepsy onset), 73 days (encompassing the time of disease onset in 70% of rats), and 7.5 months (chronic epilepsy). Data represent mean ± SEM; n = 7 sham; n = 5 epileptic (Epi); n = 5 nonepileptic (Nonepi) rats. Dot plots are shown in Supplemental Figure 10 . ** P

    Article Snippet: Supernatant HMGB1 was immunoprecipitated with 5 μg rabbit anti-HMGB1 (ab18256; Abcam) for 16 hours at 4°C as previously described ( ).

    Techniques:

    The effects of pannexin-1 inhibitors and P2X7 antagonists on corticosterone-induced HMGB1 release in primary cultured cortical astrocytes. ( A ) Astrocytes were pretreated with 10 μM carbenoxolone (CBX) for 30 min, and subsequently treated with 1 μM corticosterone for 24 h. The amount of HMGB1 in the media was quantified by Western blotting. Representative blots are shown (HMGB1: 30 kDa). The data are expressed as the mean ± SEM. ** p

    Journal: Cells

    Article Title: Corticosterone Induces HMGB1 Release in Primary Cultured Rat Cortical Astrocytes: Involvement of Pannexin-1 and P2X7 Receptor-Dependent Mechanisms

    doi: 10.3390/cells9051068

    Figure Lengend Snippet: The effects of pannexin-1 inhibitors and P2X7 antagonists on corticosterone-induced HMGB1 release in primary cultured cortical astrocytes. ( A ) Astrocytes were pretreated with 10 μM carbenoxolone (CBX) for 30 min, and subsequently treated with 1 μM corticosterone for 24 h. The amount of HMGB1 in the media was quantified by Western blotting. Representative blots are shown (HMGB1: 30 kDa). The data are expressed as the mean ± SEM. ** p

    Article Snippet: Cells were then rinsed with PBS, incubated in a blocking solution of 10% goat serum, 3% BSA, 0.2% Triton X and 0.2% Tween-20 in PBS for 30 min at 24–26 °C, and then incubated with a polyclonal antibody against HMGB1 (catalog # ab18256, 1:1000, Abcam, Cambridge, UK) for three days at 4 °C.

    Techniques: Cell Culture, Western Blot

    The effect of dexamethasone on HMGB1 release in primary cultured cortical astrocytes. ( A ) Increasing concentrations of dexamethasone induced HMGB1 release from astrocytes. Cells were treated with the indicated concentrations of dexamethasone for 24 h, and the amount of HMGB1 in the media was analyzed by Western blotting. Representative blots are shown (HMGB1: 30 kDa). The data are expressed as the mean ± SEM. * p

    Journal: Cells

    Article Title: Corticosterone Induces HMGB1 Release in Primary Cultured Rat Cortical Astrocytes: Involvement of Pannexin-1 and P2X7 Receptor-Dependent Mechanisms

    doi: 10.3390/cells9051068

    Figure Lengend Snippet: The effect of dexamethasone on HMGB1 release in primary cultured cortical astrocytes. ( A ) Increasing concentrations of dexamethasone induced HMGB1 release from astrocytes. Cells were treated with the indicated concentrations of dexamethasone for 24 h, and the amount of HMGB1 in the media was analyzed by Western blotting. Representative blots are shown (HMGB1: 30 kDa). The data are expressed as the mean ± SEM. * p

    Article Snippet: Cells were then rinsed with PBS, incubated in a blocking solution of 10% goat serum, 3% BSA, 0.2% Triton X and 0.2% Tween-20 in PBS for 30 min at 24–26 °C, and then incubated with a polyclonal antibody against HMGB1 (catalog # ab18256, 1:1000, Abcam, Cambridge, UK) for three days at 4 °C.

    Techniques: Cell Culture, Western Blot

    Effects of glucocorticoid receptor antagonist on corticosterone or dexamethasone-induced HMGB1 release in primary cultured cortical astrocytes. ( A ) Astrocytes were pretreated with 0.5 μM mifepristone (mife) for 30 min, and subsequently treated with 1 μM corticosterone for 24 h. The amount of HMGB1 in the media was quantified by Western blotting. Representative blots are shown (HMGB1: 30 kDa). The data are expressed as the mean ± S.E.M. ** p

    Journal: Cells

    Article Title: Corticosterone Induces HMGB1 Release in Primary Cultured Rat Cortical Astrocytes: Involvement of Pannexin-1 and P2X7 Receptor-Dependent Mechanisms

    doi: 10.3390/cells9051068

    Figure Lengend Snippet: Effects of glucocorticoid receptor antagonist on corticosterone or dexamethasone-induced HMGB1 release in primary cultured cortical astrocytes. ( A ) Astrocytes were pretreated with 0.5 μM mifepristone (mife) for 30 min, and subsequently treated with 1 μM corticosterone for 24 h. The amount of HMGB1 in the media was quantified by Western blotting. Representative blots are shown (HMGB1: 30 kDa). The data are expressed as the mean ± S.E.M. ** p

    Article Snippet: Cells were then rinsed with PBS, incubated in a blocking solution of 10% goat serum, 3% BSA, 0.2% Triton X and 0.2% Tween-20 in PBS for 30 min at 24–26 °C, and then incubated with a polyclonal antibody against HMGB1 (catalog # ab18256, 1:1000, Abcam, Cambridge, UK) for three days at 4 °C.

    Techniques: Cell Culture, Western Blot

    The effect of corticosterone on HMGB1 mRNA and intracellular HMGB1 protein expression in primary cultured cortical astrocytes. ( A ) Astrocytes were treated with 1 μM corticosterone for the indicated periods of time or the indicated concentrations of corticosterone for 24 h. HMGB1 mRNA expression (ratio of HMGB1 mRNA: GAPDH mRNA) was quantified by real time PCR. The data are expressed as the mean ± SEM. ( n = 3–5). ( B ) Astrocytes were treated with 1 μM corticosterone for the indicated periods of time or the indicated concentrations of corticosterone for 24 h. HMGB1 protein was quantified by Western blotting, and representative blots are shown (HMGB1: 30 kDa, β-actin: 42 kDa). The data are expressed as the mean ± SEM. ( n = 3–5).

    Journal: Cells

    Article Title: Corticosterone Induces HMGB1 Release in Primary Cultured Rat Cortical Astrocytes: Involvement of Pannexin-1 and P2X7 Receptor-Dependent Mechanisms

    doi: 10.3390/cells9051068

    Figure Lengend Snippet: The effect of corticosterone on HMGB1 mRNA and intracellular HMGB1 protein expression in primary cultured cortical astrocytes. ( A ) Astrocytes were treated with 1 μM corticosterone for the indicated periods of time or the indicated concentrations of corticosterone for 24 h. HMGB1 mRNA expression (ratio of HMGB1 mRNA: GAPDH mRNA) was quantified by real time PCR. The data are expressed as the mean ± SEM. ( n = 3–5). ( B ) Astrocytes were treated with 1 μM corticosterone for the indicated periods of time or the indicated concentrations of corticosterone for 24 h. HMGB1 protein was quantified by Western blotting, and representative blots are shown (HMGB1: 30 kDa, β-actin: 42 kDa). The data are expressed as the mean ± SEM. ( n = 3–5).

    Article Snippet: Cells were then rinsed with PBS, incubated in a blocking solution of 10% goat serum, 3% BSA, 0.2% Triton X and 0.2% Tween-20 in PBS for 30 min at 24–26 °C, and then incubated with a polyclonal antibody against HMGB1 (catalog # ab18256, 1:1000, Abcam, Cambridge, UK) for three days at 4 °C.

    Techniques: Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Western Blot

    Effects of corticosterone on intracellular localization of HMGB1 in primary cultured cortical astrocytes. ( A ) Immunocytochemistry of HMGB1 in rat primary cultured cortical astrocytes. Corticosterone-treated cells (CORT; 1 μM, 24 h) exhibited subcellular HMGB1 expression compared with vehicle. Images a, b and c, or d, e and f, are from the same fields, respectively. Scale bar = 20 μm. ( B ) The percentage of nuclear staining of HMGB1 to total DAPI positive cells decreased after corticosterone treatment. The data are expressed as the mean ± SEM. * p

    Journal: Cells

    Article Title: Corticosterone Induces HMGB1 Release in Primary Cultured Rat Cortical Astrocytes: Involvement of Pannexin-1 and P2X7 Receptor-Dependent Mechanisms

    doi: 10.3390/cells9051068

    Figure Lengend Snippet: Effects of corticosterone on intracellular localization of HMGB1 in primary cultured cortical astrocytes. ( A ) Immunocytochemistry of HMGB1 in rat primary cultured cortical astrocytes. Corticosterone-treated cells (CORT; 1 μM, 24 h) exhibited subcellular HMGB1 expression compared with vehicle. Images a, b and c, or d, e and f, are from the same fields, respectively. Scale bar = 20 μm. ( B ) The percentage of nuclear staining of HMGB1 to total DAPI positive cells decreased after corticosterone treatment. The data are expressed as the mean ± SEM. * p

    Article Snippet: Cells were then rinsed with PBS, incubated in a blocking solution of 10% goat serum, 3% BSA, 0.2% Triton X and 0.2% Tween-20 in PBS for 30 min at 24–26 °C, and then incubated with a polyclonal antibody against HMGB1 (catalog # ab18256, 1:1000, Abcam, Cambridge, UK) for three days at 4 °C.

    Techniques: Cell Culture, Immunocytochemistry, Expressing, Staining

    The effect of corticosterone on high-mobility group box-1 (HMGB1) release in primary cultured cortical astrocytes. ( A ) Increasing concentrations of corticosterone induced HMGB1 release from astrocytes. Cells were treated with the indicated concentrations of corticosterone for 24 h, and the amount of HMGB1 in the media was analyzed by Western blotting. Representative blots are shown (HMGB1: 30 kDa). The data are expressed as the mean ± SEM. * p

    Journal: Cells

    Article Title: Corticosterone Induces HMGB1 Release in Primary Cultured Rat Cortical Astrocytes: Involvement of Pannexin-1 and P2X7 Receptor-Dependent Mechanisms

    doi: 10.3390/cells9051068

    Figure Lengend Snippet: The effect of corticosterone on high-mobility group box-1 (HMGB1) release in primary cultured cortical astrocytes. ( A ) Increasing concentrations of corticosterone induced HMGB1 release from astrocytes. Cells were treated with the indicated concentrations of corticosterone for 24 h, and the amount of HMGB1 in the media was analyzed by Western blotting. Representative blots are shown (HMGB1: 30 kDa). The data are expressed as the mean ± SEM. * p

    Article Snippet: Cells were then rinsed with PBS, incubated in a blocking solution of 10% goat serum, 3% BSA, 0.2% Triton X and 0.2% Tween-20 in PBS for 30 min at 24–26 °C, and then incubated with a polyclonal antibody against HMGB1 (catalog # ab18256, 1:1000, Abcam, Cambridge, UK) for three days at 4 °C.

    Techniques: Cell Culture, Western Blot

    Interference of HMGB1 expression with siRNA

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: High-mobility group box 1: A novel target for treatment of Pseudomonas aeruginosa keratitis

    doi: 10.4049/jimmunol.1401684

    Figure Lengend Snippet: Interference of HMGB1 expression with siRNA

    Article Snippet: A 50 µl aliquot of each supernatant was assayed in duplicate for HMGB1 protein per the manufacturer’s instructions (Chondrex, Inc. Redmond, WA).

    Techniques: Expressing

    HMGB1 neutralization

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: High-mobility group box 1: A novel target for treatment of Pseudomonas aeruginosa keratitis

    doi: 10.4049/jimmunol.1401684

    Figure Lengend Snippet: HMGB1 neutralization

    Article Snippet: A 50 µl aliquot of each supernatant was assayed in duplicate for HMGB1 protein per the manufacturer’s instructions (Chondrex, Inc. Redmond, WA).

    Techniques: Neutralization

    HMGB1 corneal staining after VIP treatment

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: High-mobility group box 1: A novel target for treatment of Pseudomonas aeruginosa keratitis

    doi: 10.4049/jimmunol.1401684

    Figure Lengend Snippet: HMGB1 corneal staining after VIP treatment

    Article Snippet: A 50 µl aliquot of each supernatant was assayed in duplicate for HMGB1 protein per the manufacturer’s instructions (Chondrex, Inc. Redmond, WA).

    Techniques: Staining

    HMGB1 knockdown-induced apoptosis of RCC cells. Notes: ( A ) Apoptosis in A498 cells transfected with HMGB1-shRNA, Con-shRNA, and the indicated vectors were analyzed by flow cytometry. ( B ) Apoptosis in ACHN cells transfected with HMGB1-siRNA, Con-siRNA, and the indicated vectors were analyzed by flow cytometry. The results are representative of 3 separate experiments. ** P

    Journal: OncoTargets and therapy

    Article Title: HMGB1/RAGE axis mediates the apoptosis, invasion, autophagy, and angiogenesis of the renal cell carcinoma

    doi: 10.2147/OTT.S167197

    Figure Lengend Snippet: HMGB1 knockdown-induced apoptosis of RCC cells. Notes: ( A ) Apoptosis in A498 cells transfected with HMGB1-shRNA, Con-shRNA, and the indicated vectors were analyzed by flow cytometry. ( B ) Apoptosis in ACHN cells transfected with HMGB1-siRNA, Con-siRNA, and the indicated vectors were analyzed by flow cytometry. The results are representative of 3 separate experiments. ** P

    Article Snippet: In addition, plasmid pcDNA3.1-HMGB1 was transfected into RCC cells to upregulate the expression of HMGB1 ( ).

    Techniques: Transfection, shRNA, Flow Cytometry, Cytometry

    HMGB1 knockdown inhibited RCC tumor growth in vivo. Notes: ( A ) The A498 xenografts were intratumorally injected with HMGB1 control or siRNA2 (twice a week), and the tumor volumes were measured using caliper (weekly). ( B , C ) The mice were sacrificed using with CO 2 after 4 weeks of treatments, and the tumors were isolated and weighted. ( D ) The body weights of mice were monitored weekly. ** P

    Journal: OncoTargets and therapy

    Article Title: HMGB1/RAGE axis mediates the apoptosis, invasion, autophagy, and angiogenesis of the renal cell carcinoma

    doi: 10.2147/OTT.S167197

    Figure Lengend Snippet: HMGB1 knockdown inhibited RCC tumor growth in vivo. Notes: ( A ) The A498 xenografts were intratumorally injected with HMGB1 control or siRNA2 (twice a week), and the tumor volumes were measured using caliper (weekly). ( B , C ) The mice were sacrificed using with CO 2 after 4 weeks of treatments, and the tumors were isolated and weighted. ( D ) The body weights of mice were monitored weekly. ** P

    Article Snippet: In addition, plasmid pcDNA3.1-HMGB1 was transfected into RCC cells to upregulate the expression of HMGB1 ( ).

    Techniques: In Vivo, Injection, Mouse Assay, Isolation

    The effects of HMGB1 on RCC-stimulated VEGF and VEGFR2 expressions in HUVEC. Notes: ( A , B ) The effects of HMGB1/sRAGE on RCC-stimulated VEGF and VEGFR mRNA expressions in HUVEC were analyzed by qRT-PCR. ( C , D ) The effects of HMGB1/sRAGE on RCC-stimulated VEGF and VEGFR2 proteins expression in HUVEC were detected by Western blot analysis. * P

    Journal: OncoTargets and therapy

    Article Title: HMGB1/RAGE axis mediates the apoptosis, invasion, autophagy, and angiogenesis of the renal cell carcinoma

    doi: 10.2147/OTT.S167197

    Figure Lengend Snippet: The effects of HMGB1 on RCC-stimulated VEGF and VEGFR2 expressions in HUVEC. Notes: ( A , B ) The effects of HMGB1/sRAGE on RCC-stimulated VEGF and VEGFR mRNA expressions in HUVEC were analyzed by qRT-PCR. ( C , D ) The effects of HMGB1/sRAGE on RCC-stimulated VEGF and VEGFR2 proteins expression in HUVEC were detected by Western blot analysis. * P

    Article Snippet: In addition, plasmid pcDNA3.1-HMGB1 was transfected into RCC cells to upregulate the expression of HMGB1 ( ).

    Techniques: Quantitative RT-PCR, Expressing, Western Blot

    HMGB1 regulated RAGE and autophagic protein expressions in human RCC cell lines. Notes: ( A ) The expression and quantification of HMGB1, RAGE, LC3, and Beclin1 in the A498 cell line were analyzed by Western blot analysis. ( B ) The expression and quantification of HMGB1, RAGE, LC3, and Beclin1 in the ACHN cell line were analyzed by qRT-PCR. Data are presented as mean ± SD from three independent experiments. * P

    Journal: OncoTargets and therapy

    Article Title: HMGB1/RAGE axis mediates the apoptosis, invasion, autophagy, and angiogenesis of the renal cell carcinoma

    doi: 10.2147/OTT.S167197

    Figure Lengend Snippet: HMGB1 regulated RAGE and autophagic protein expressions in human RCC cell lines. Notes: ( A ) The expression and quantification of HMGB1, RAGE, LC3, and Beclin1 in the A498 cell line were analyzed by Western blot analysis. ( B ) The expression and quantification of HMGB1, RAGE, LC3, and Beclin1 in the ACHN cell line were analyzed by qRT-PCR. Data are presented as mean ± SD from three independent experiments. * P

    Article Snippet: In addition, plasmid pcDNA3.1-HMGB1 was transfected into RCC cells to upregulate the expression of HMGB1 ( ).

    Techniques: Expressing, Western Blot, Quantitative RT-PCR

    HMGB1 knockdown decreased the viability of A498 and ACHN cells. Notes: ( A ) The RAGE and HMGB1 proteins in RCC cell lines were detected by Western blot. ( B ) The cells were treated with control siRNA, HMGB1 siRNA1, siRNA2, or siRNA3 for 48 h, and then, HMGB1 mRNA levels in cells were tested by qRT-PCR. ( C ) A498 and ACHN cells were transfected with pcDNA3.1-HMGB1 plasmid or control, and then, HMGB1 mRNA levels in cells were tested by qRT-PCR after 48 h incubation. ( D ) The A498 and ACHN cells were transfected with control siRNA, HMGB1 siRNA-3 HMGB1-Vec, or sRAGE were seeded into the 96-well plate, then cell proliferation was measured by CCK8 assay. Data are presented as mean ± SD from three independent experiments. * P

    Journal: OncoTargets and therapy

    Article Title: HMGB1/RAGE axis mediates the apoptosis, invasion, autophagy, and angiogenesis of the renal cell carcinoma

    doi: 10.2147/OTT.S167197

    Figure Lengend Snippet: HMGB1 knockdown decreased the viability of A498 and ACHN cells. Notes: ( A ) The RAGE and HMGB1 proteins in RCC cell lines were detected by Western blot. ( B ) The cells were treated with control siRNA, HMGB1 siRNA1, siRNA2, or siRNA3 for 48 h, and then, HMGB1 mRNA levels in cells were tested by qRT-PCR. ( C ) A498 and ACHN cells were transfected with pcDNA3.1-HMGB1 plasmid or control, and then, HMGB1 mRNA levels in cells were tested by qRT-PCR after 48 h incubation. ( D ) The A498 and ACHN cells were transfected with control siRNA, HMGB1 siRNA-3 HMGB1-Vec, or sRAGE were seeded into the 96-well plate, then cell proliferation was measured by CCK8 assay. Data are presented as mean ± SD from three independent experiments. * P

    Article Snippet: In addition, plasmid pcDNA3.1-HMGB1 was transfected into RCC cells to upregulate the expression of HMGB1 ( ).

    Techniques: Western Blot, Quantitative RT-PCR, Transfection, Plasmid Preparation, Incubation, CCK-8 Assay

    HMGB1 knockdown suppressed the invasion of RCC cells. Notes: ( A ) The metastatic ability of A498 cells transfected with HMGB1-shRNA, Con-shRNA, and the indicated vectors were analyzed by Matrigel invasion assays. ( B ) The number of A498 cells that invaded through the Matrigel-coated membrane. ( C ) The metastatic ability of ACHN cells transfected with HMGB1-siRNA, Con-siRNA, and the indicated vectors were analyzed by Matrigel invasion assays. ( D ) The number of ACHN cells that invaded through the Matrigel-coated membrane. Data are presented as mean ± SD from 3 independent experiments. * P

    Journal: OncoTargets and therapy

    Article Title: HMGB1/RAGE axis mediates the apoptosis, invasion, autophagy, and angiogenesis of the renal cell carcinoma

    doi: 10.2147/OTT.S167197

    Figure Lengend Snippet: HMGB1 knockdown suppressed the invasion of RCC cells. Notes: ( A ) The metastatic ability of A498 cells transfected with HMGB1-shRNA, Con-shRNA, and the indicated vectors were analyzed by Matrigel invasion assays. ( B ) The number of A498 cells that invaded through the Matrigel-coated membrane. ( C ) The metastatic ability of ACHN cells transfected with HMGB1-siRNA, Con-siRNA, and the indicated vectors were analyzed by Matrigel invasion assays. ( D ) The number of ACHN cells that invaded through the Matrigel-coated membrane. Data are presented as mean ± SD from 3 independent experiments. * P

    Article Snippet: In addition, plasmid pcDNA3.1-HMGB1 was transfected into RCC cells to upregulate the expression of HMGB1 ( ).

    Techniques: Transfection, shRNA

    Effect of JNK inhibition on TNF-α or TNF-α/HMGB1-induced myocyte apoptosis. Cardiomyocytes were pretreated with the JNK inhibitor (SP-600125; 10 μM) for 30 min and subsequently exposed to M199 or M199 containing either TNF-α (40 ng/ml) or TNF-α/HMGB1 (1 μg/ml) cocktail. After a 24-h incubation period, cardiomyocyte apoptosis was assessed with caspase-3 activity ( top ) and a cell death ELISA kit ( bottom ). Values are means ± SE; n = 3. * P

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    Article Title: Endogenous HMGB1 contributes to ischemia-reperfusion-induced myocardial apoptosis by potentiating the effect of TNF-?/JNK

    doi: 10.1152/ajpheart.00703.2010

    Figure Lengend Snippet: Effect of JNK inhibition on TNF-α or TNF-α/HMGB1-induced myocyte apoptosis. Cardiomyocytes were pretreated with the JNK inhibitor (SP-600125; 10 μM) for 30 min and subsequently exposed to M199 or M199 containing either TNF-α (40 ng/ml) or TNF-α/HMGB1 (1 μg/ml) cocktail. After a 24-h incubation period, cardiomyocyte apoptosis was assessed with caspase-3 activity ( top ) and a cell death ELISA kit ( bottom ). Values are means ± SE; n = 3. * P

    Article Snippet: Collagenase type II was from Worthington Biochemical, Lakewood, NJ; recombinant full length HMGB1 and anti-mouse HMGB1 polyclonal antibody were from ABCAM, Cambridge, MA (catalog no. ab18256); polyvinylidene difluoride membrane was from Bio-Rad Laboratories, Mississauga, ON; ECL plus Western blotting detection kit was from GE Healthcare, Mississauga, ON; HMGB1 ELISA kit was from Shino-Test; mouse TNF-α ELISA set was from BD Biosciences; antibodies against phosphorylated and total JNK (catalog nos.

    Techniques: Inhibition, Incubation, Activity Assay, Enzyme-linked Immunosorbent Assay

    Interaction of HMGB1 and TNF-α in the A/R-induced myocyte apoptosis. A : HMGB1 was incubated with isolated cardiomyocytes at the doses indicated for 24 h, and apoptosis was assessed with caspase-3 activity and a cell death ELISA kit. n = 3. B : cardiomyocytes were challenged with N/R or A/R, with or without A-box (10 μg/ml), which was added to the myocytes 2 h after reoxygenation. Releasing of TNF-α into the supernatant was measured 48 h after reoxygenation using a mouse TNF-α ELISA assay set. n = 3. * P

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    Article Title: Endogenous HMGB1 contributes to ischemia-reperfusion-induced myocardial apoptosis by potentiating the effect of TNF-?/JNK

    doi: 10.1152/ajpheart.00703.2010

    Figure Lengend Snippet: Interaction of HMGB1 and TNF-α in the A/R-induced myocyte apoptosis. A : HMGB1 was incubated with isolated cardiomyocytes at the doses indicated for 24 h, and apoptosis was assessed with caspase-3 activity and a cell death ELISA kit. n = 3. B : cardiomyocytes were challenged with N/R or A/R, with or without A-box (10 μg/ml), which was added to the myocytes 2 h after reoxygenation. Releasing of TNF-α into the supernatant was measured 48 h after reoxygenation using a mouse TNF-α ELISA assay set. n = 3. * P

    Article Snippet: Collagenase type II was from Worthington Biochemical, Lakewood, NJ; recombinant full length HMGB1 and anti-mouse HMGB1 polyclonal antibody were from ABCAM, Cambridge, MA (catalog no. ab18256); polyvinylidene difluoride membrane was from Bio-Rad Laboratories, Mississauga, ON; ECL plus Western blotting detection kit was from GE Healthcare, Mississauga, ON; HMGB1 ELISA kit was from Shino-Test; mouse TNF-α ELISA set was from BD Biosciences; antibodies against phosphorylated and total JNK (catalog nos.

    Techniques: Incubation, Isolation, Activity Assay, Enzyme-linked Immunosorbent Assay

    Serum HMGB1 levels are increased in colorectal cancer patients. The sera of 219 cancer patients were screened using HMGB1 ELISA, and the findings were compared with those of 75 non-cancerous healthy controls. Serum CEA levels were also measured in both groups. Each serum value was transferred to natural logarithm to draw a data comparison plot. (A) Serum HMGB1 levels were 1.5-fold higher in cancer patients than in healthy controls (the mean serum concentrations were 58.8±126.2 ng/mL in colorectal cancer patients and 39.7±16.2 ng/mL in control subjects). The P-value was calculated by the Welch's t-test ( = 0.03) (B) Serum CEA levels were elevated in cancer patients compared to those in healthy controls (the mean serum concentrations were 18.3±100.8 ng/mL in patients with colorectal carcinoma and 1.9±1.8 ng/mL in control subjects). The P-value was calculated by the Welch's t-test ( = 0.02) (C) HMGB1 concentrations were depicted according to different tumor stages. (D) CEA concentrations were depicted according to different tumor stages. CEA levels were elevated in advanced tumor stages.

    Journal: PLoS ONE

    Article Title: Diagnostic Significance of Serum HMGB1 in Colorectal Carcinomas

    doi: 10.1371/journal.pone.0034318

    Figure Lengend Snippet: Serum HMGB1 levels are increased in colorectal cancer patients. The sera of 219 cancer patients were screened using HMGB1 ELISA, and the findings were compared with those of 75 non-cancerous healthy controls. Serum CEA levels were also measured in both groups. Each serum value was transferred to natural logarithm to draw a data comparison plot. (A) Serum HMGB1 levels were 1.5-fold higher in cancer patients than in healthy controls (the mean serum concentrations were 58.8±126.2 ng/mL in colorectal cancer patients and 39.7±16.2 ng/mL in control subjects). The P-value was calculated by the Welch's t-test ( = 0.03) (B) Serum CEA levels were elevated in cancer patients compared to those in healthy controls (the mean serum concentrations were 18.3±100.8 ng/mL in patients with colorectal carcinoma and 1.9±1.8 ng/mL in control subjects). The P-value was calculated by the Welch's t-test ( = 0.02) (C) HMGB1 concentrations were depicted according to different tumor stages. (D) CEA concentrations were depicted according to different tumor stages. CEA levels were elevated in advanced tumor stages.

    Article Snippet: ELISA Serum concentrations of HMGB1 were evaluated by ELISA using the HMGB1 ELISA Kit II (Shino-test, Tokyo, Japan).

    Techniques: Enzyme-linked Immunosorbent Assay

    Survival curves of high-mobility group box 1 and vascular endothelial growth factor C in intrahepatic cholangiocarcinoma. Kaplan-Meier method univariate analyses of HMGB1 (A); VEGF-C (B); Survival curves of intrahepatic cholangiocarcinoma patients with

    Journal: World Journal of Gastroenterology : WJG

    Article Title: High-mobility group box 1 expression and lymph node metastasis in intrahepatic cholangiocarcinoma

    doi: 10.3748/wjg.v21.i11.3256

    Figure Lengend Snippet: Survival curves of high-mobility group box 1 and vascular endothelial growth factor C in intrahepatic cholangiocarcinoma. Kaplan-Meier method univariate analyses of HMGB1 (A); VEGF-C (B); Survival curves of intrahepatic cholangiocarcinoma patients with

    Article Snippet: Spearman’s Rank correlation coefficient was used to identify the correlation between HMGB1 and LMVD.

    Techniques:

    Expression of HMGB1 and VEGF-C and correlations with clinicopathologic parameters

    Journal: World Journal of Gastroenterology : WJG

    Article Title: High-mobility group box 1 expression and lymph node metastasis in intrahepatic cholangiocarcinoma

    doi: 10.3748/wjg.v21.i11.3256

    Figure Lengend Snippet: Expression of HMGB1 and VEGF-C and correlations with clinicopathologic parameters

    Article Snippet: Spearman’s Rank correlation coefficient was used to identify the correlation between HMGB1 and LMVD.

    Techniques: Expressing

    Prognostic value of HMGB1 expression in IHCC

    Journal: World Journal of Gastroenterology : WJG

    Article Title: High-mobility group box 1 expression and lymph node metastasis in intrahepatic cholangiocarcinoma

    doi: 10.3748/wjg.v21.i11.3256

    Figure Lengend Snippet: Prognostic value of HMGB1 expression in IHCC

    Article Snippet: Spearman’s Rank correlation coefficient was used to identify the correlation between HMGB1 and LMVD.

    Techniques: Expressing

    High-mobility group box 1 overexpression promotes epithelial-mesenchymal transition in RBE cell line. A: HMGB1 protein expression level in RBE, HUCCT-1, and QBC939 cell lines; B: mRNA expression level of HMGB1; C: Western blot; D: HMGB1 siRNA knockdown

    Journal: World Journal of Gastroenterology : WJG

    Article Title: High-mobility group box 1 expression and lymph node metastasis in intrahepatic cholangiocarcinoma

    doi: 10.3748/wjg.v21.i11.3256

    Figure Lengend Snippet: High-mobility group box 1 overexpression promotes epithelial-mesenchymal transition in RBE cell line. A: HMGB1 protein expression level in RBE, HUCCT-1, and QBC939 cell lines; B: mRNA expression level of HMGB1; C: Western blot; D: HMGB1 siRNA knockdown

    Article Snippet: Spearman’s Rank correlation coefficient was used to identify the correlation between HMGB1 and LMVD.

    Techniques: Over Expression, Expressing, Western Blot

    Correlations of HMGB1 with LMVD and VEGF-C

    Journal: World Journal of Gastroenterology : WJG

    Article Title: High-mobility group box 1 expression and lymph node metastasis in intrahepatic cholangiocarcinoma

    doi: 10.3748/wjg.v21.i11.3256

    Figure Lengend Snippet: Correlations of HMGB1 with LMVD and VEGF-C

    Article Snippet: Spearman’s Rank correlation coefficient was used to identify the correlation between HMGB1 and LMVD.

    Techniques:

    SAHA induced down-regulation of NF- κ B1 was HMGB1 dependent. LX2 cells were transfected by siR-NC or siR-HMGB1 48 h before 2.5 µM SAHA treatment. (A) The mRNA expression of NF- κ B1 (p50) in each group was detected by RT-PCR. (B) Protein expressions of p50 and p65 in each group were determined by western blot assay. (C) Dual luciferase reporter assay. The activity of NF- κ B in each group was detected by a dual luciferase reporter system. NF- κ B driven firefly luciferase activity was assayed by a dual luciferase reporter system, renilla luciferase activity served as internal control, the results were expressed as relative luciferase activity. The mRNA or protein expressions were normalized to GAPDH. ∗∗ P

    Journal: PeerJ

    Article Title: Suberoylanilide hydroxamic acid suppresses hepatic stellate cells activation by HMGB1 dependent reduction of NF-κB1

    doi: 10.7717/peerj.1362

    Figure Lengend Snippet: SAHA induced down-regulation of NF- κ B1 was HMGB1 dependent. LX2 cells were transfected by siR-NC or siR-HMGB1 48 h before 2.5 µM SAHA treatment. (A) The mRNA expression of NF- κ B1 (p50) in each group was detected by RT-PCR. (B) Protein expressions of p50 and p65 in each group were determined by western blot assay. (C) Dual luciferase reporter assay. The activity of NF- κ B in each group was detected by a dual luciferase reporter system. NF- κ B driven firefly luciferase activity was assayed by a dual luciferase reporter system, renilla luciferase activity served as internal control, the results were expressed as relative luciferase activity. The mRNA or protein expressions were normalized to GAPDH. ∗∗ P

    Article Snippet: LX2 cells were cultured on coverslips were fixed with 2% paraformaldehyde for 15 min on ice, followed by incubation with antibody against HMGB1 (Abcam) at 4 °C overnight, Cy3 labeled secondary antibody (Beyotime) for 1 h in dark.

    Techniques: Transfection, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Luciferase, Reporter Assay, Activity Assay

    SAHA treatment improved the lysine acetylation of intracellular HMGB1. (A) The protein level of HMGB1 in the whole cell or nuclear lysates of SAHA treated LX2 cells was detected by western blot, ns, not significant, P > 0.05. GAPDH was used as a loading control for the whole cell lysates, and PCNA were used as a loading control for the nuclear fraction. (B) The lysine acetylation levels of total proteins from SAHA-treated LX2 cells were analyzed by western blot. The concentrations of SAHA were 0.5 µM, 1.0 µM, 2.5 µM, 5.0 µM and 10.0 µM as indicated. GAPDH was used as a loading control. (C) Lysine acetylation of HMGB1 was detected by immunoprecipitation. IgG was served as a negative control.

    Journal: PeerJ

    Article Title: Suberoylanilide hydroxamic acid suppresses hepatic stellate cells activation by HMGB1 dependent reduction of NF-κB1

    doi: 10.7717/peerj.1362

    Figure Lengend Snippet: SAHA treatment improved the lysine acetylation of intracellular HMGB1. (A) The protein level of HMGB1 in the whole cell or nuclear lysates of SAHA treated LX2 cells was detected by western blot, ns, not significant, P > 0.05. GAPDH was used as a loading control for the whole cell lysates, and PCNA were used as a loading control for the nuclear fraction. (B) The lysine acetylation levels of total proteins from SAHA-treated LX2 cells were analyzed by western blot. The concentrations of SAHA were 0.5 µM, 1.0 µM, 2.5 µM, 5.0 µM and 10.0 µM as indicated. GAPDH was used as a loading control. (C) Lysine acetylation of HMGB1 was detected by immunoprecipitation. IgG was served as a negative control.

    Article Snippet: LX2 cells were cultured on coverslips were fixed with 2% paraformaldehyde for 15 min on ice, followed by incubation with antibody against HMGB1 (Abcam) at 4 °C overnight, Cy3 labeled secondary antibody (Beyotime) for 1 h in dark.

    Techniques: Western Blot, Immunoprecipitation, Negative Control

    Membrane attack complex (MAC) deposition is reduced by high-mobility group box 1 (HMGB1) neutralizing antibody (Ab) in middle cerebral arterial occlusion (MCAO) ischemia mouse model. (A) HMGB1 staining of ICR mouse brain 4 h after MCAO. Contralateral and ipsilateral brain sections were stained for HMGB1 and collagen (Col) IV, which visualizes most blood vessels and capillaries. The images captured at low and high magnifications are shown. (B) Pearson’s coefficient for the overlapping of HMGB1 and DAPI in low magnification of three fields was calculated. Error bars are mean ± SD. ** P

    Journal: Frontiers in Immunology

    Article Title: High-Mobility Group Box 1-Induced Complement Activation Causes Sterile Inflammation

    doi: 10.3389/fimmu.2018.00705

    Figure Lengend Snippet: Membrane attack complex (MAC) deposition is reduced by high-mobility group box 1 (HMGB1) neutralizing antibody (Ab) in middle cerebral arterial occlusion (MCAO) ischemia mouse model. (A) HMGB1 staining of ICR mouse brain 4 h after MCAO. Contralateral and ipsilateral brain sections were stained for HMGB1 and collagen (Col) IV, which visualizes most blood vessels and capillaries. The images captured at low and high magnifications are shown. (B) Pearson’s coefficient for the overlapping of HMGB1 and DAPI in low magnification of three fields was calculated. Error bars are mean ± SD. ** P

    Article Snippet: Rabbit anti-HMGB1 Ab (1:1,500, Abcam #18256) was added for 1 h at RT after washing.

    Techniques: Staining

    High-mobility group box 1 (HMGB1)-induced complement activation model. HMGB1 protein is actively secreted or passively released by stress injury on cells or organs in the forms of three different redox statuses, which may differently modulate immunological activities. Extracellular HMGB1 can activate the classical pathway of complement system in an antibody (Ab)-independent manner after binding to C1q, resulting in forming C5b-9 membrane attack complexes (MAC) where HMGB1 is accumulated. Thus, HMGB1-induced complement activation is proposed to be able to exacerbate sterile inflammation.

    Journal: Frontiers in Immunology

    Article Title: High-Mobility Group Box 1-Induced Complement Activation Causes Sterile Inflammation

    doi: 10.3389/fimmu.2018.00705

    Figure Lengend Snippet: High-mobility group box 1 (HMGB1)-induced complement activation model. HMGB1 protein is actively secreted or passively released by stress injury on cells or organs in the forms of three different redox statuses, which may differently modulate immunological activities. Extracellular HMGB1 can activate the classical pathway of complement system in an antibody (Ab)-independent manner after binding to C1q, resulting in forming C5b-9 membrane attack complexes (MAC) where HMGB1 is accumulated. Thus, HMGB1-induced complement activation is proposed to be able to exacerbate sterile inflammation.

    Article Snippet: Rabbit anti-HMGB1 Ab (1:1,500, Abcam #18256) was added for 1 h at RT after washing.

    Techniques: Activation Assay, Binding Assay

    High-mobility group box 1 (HMGB1)-mediated cleavage of C4 and C3 and the formation of C5b-9 [membrane attack complex (MAC)]. (A) Measurement of C4b. A microtiter plate was coated with Euk-HMGB1 (10 µg/ml, R D) and incubated with various concentrations of normal human serum (NHS) to test the deposition of C4b using an enzyme-linked immunosorbent assay (ELISA) (left). Human IgG (10 µg/ml) was used as the positive control (right). (B) To determine C3a and iC3b, the cleavage product of C3b. Fifty microliters of NHS (diluted 1:5) was incubated with increasing amounts of HMGB1 for 30 min at 37°C, then performed Western blot analysis using anti-C3/iC3b or anti-C3a antibodies. Aggregated human IgG and Ig-free BSA served as the positive and negative controls, respectively. Ig light chain (IgL) was used as input control. (C,D) Measurements of complement activation products of C3c, the cleaved form of C3b, and C5b-9. Biotinylated HMGB1s were incubated with streptavidin-coated microspheres in 10% NHS, which was pre-absorbed with non-coated microspheres, for 30 min at 37°C. Immunofluorescence staining was performed with anti-HMGB1 and fluorescein isothiocyanate-conjugated anti-C3c antibody (Ab). For C5b-9 deposition, immunofluorescence staining was performed with anti-HMGB1 (green) and anti-C5b-9 Ab (red). Biotinylated human IgG was used as the positive control. (E) A microtiter plate was coated with HMGB1 (10 µg/ml) and incubated with various concentrations of NHS to test the deposition of C5b-9 using an ELISA. Aggregated human IgG was used as the positive control. (F) C1q-dependent MAC formation. A microtiter plate was coated with HMGB1 (10 µg/ml) and incubated with various concentrations of NHS or C1q-depleted human serum (C1q-dep HS) to test the deposition of MAC formation using an ELISA for C5b-9. To restore the effect of C1q, 20 µg/ml of C1q was added to C1q-depleted human serum. ns: not significant. (G,H) Measurement of complement activation after HMGB1 injection in mice. C57BL/6 mice were intravenously injected with 100 µg of HMGB1 or PBS to observe if HMGB1 can activate complement in vivo ( N = 3 per group). Blood samples were collected at 0, 30, and 90 min after the injection. iC3b (arrow) was detected using Western blot analysis of serum sample from one representative mouse. Relative band intensity of iC3b was analyzed. All data shown here are representative of at least three independent experiments with similar results. Error bars are mean ± SD. * P

    Journal: Frontiers in Immunology

    Article Title: High-Mobility Group Box 1-Induced Complement Activation Causes Sterile Inflammation

    doi: 10.3389/fimmu.2018.00705

    Figure Lengend Snippet: High-mobility group box 1 (HMGB1)-mediated cleavage of C4 and C3 and the formation of C5b-9 [membrane attack complex (MAC)]. (A) Measurement of C4b. A microtiter plate was coated with Euk-HMGB1 (10 µg/ml, R D) and incubated with various concentrations of normal human serum (NHS) to test the deposition of C4b using an enzyme-linked immunosorbent assay (ELISA) (left). Human IgG (10 µg/ml) was used as the positive control (right). (B) To determine C3a and iC3b, the cleavage product of C3b. Fifty microliters of NHS (diluted 1:5) was incubated with increasing amounts of HMGB1 for 30 min at 37°C, then performed Western blot analysis using anti-C3/iC3b or anti-C3a antibodies. Aggregated human IgG and Ig-free BSA served as the positive and negative controls, respectively. Ig light chain (IgL) was used as input control. (C,D) Measurements of complement activation products of C3c, the cleaved form of C3b, and C5b-9. Biotinylated HMGB1s were incubated with streptavidin-coated microspheres in 10% NHS, which was pre-absorbed with non-coated microspheres, for 30 min at 37°C. Immunofluorescence staining was performed with anti-HMGB1 and fluorescein isothiocyanate-conjugated anti-C3c antibody (Ab). For C5b-9 deposition, immunofluorescence staining was performed with anti-HMGB1 (green) and anti-C5b-9 Ab (red). Biotinylated human IgG was used as the positive control. (E) A microtiter plate was coated with HMGB1 (10 µg/ml) and incubated with various concentrations of NHS to test the deposition of C5b-9 using an ELISA. Aggregated human IgG was used as the positive control. (F) C1q-dependent MAC formation. A microtiter plate was coated with HMGB1 (10 µg/ml) and incubated with various concentrations of NHS or C1q-depleted human serum (C1q-dep HS) to test the deposition of MAC formation using an ELISA for C5b-9. To restore the effect of C1q, 20 µg/ml of C1q was added to C1q-depleted human serum. ns: not significant. (G,H) Measurement of complement activation after HMGB1 injection in mice. C57BL/6 mice were intravenously injected with 100 µg of HMGB1 or PBS to observe if HMGB1 can activate complement in vivo ( N = 3 per group). Blood samples were collected at 0, 30, and 90 min after the injection. iC3b (arrow) was detected using Western blot analysis of serum sample from one representative mouse. Relative band intensity of iC3b was analyzed. All data shown here are representative of at least three independent experiments with similar results. Error bars are mean ± SD. * P

    Article Snippet: Rabbit anti-HMGB1 Ab (1:1,500, Abcam #18256) was added for 1 h at RT after washing.

    Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Positive Control, Western Blot, Activation Assay, Immunofluorescence, Staining, Injection, Mouse Assay, In Vivo

    High-mobility group box 1 (HMGB1) B box activates complement cascade. (A) Schematic overview of recombinant HMGB1 proteins used: wild-type (WT) HMGB1, boxes A (aa 1-79) and B (aa 88-162), ΔC-HMGB1 (aa 1-185), and ΔN-HMGB1 (aa 11-215). (B,C) Complement consumption assessment. WT HMGB1 proteins (B) or HMGB1 variants (C) were incubated with the diluted normal human serum (NHS) containing CH 50 activity in GVB 2+ buffer for 30 min at 37°C. After complement consumption by HMGB1, sRBCs were added in the consumed NHS for 30 min at 37°C. Aggregated human IgG and IgG-free BSA (each 30 µg/ml) were used as the positive and negative controls, respectively. Data shown are the mean ± SD of three independent repeats. * P

    Journal: Frontiers in Immunology

    Article Title: High-Mobility Group Box 1-Induced Complement Activation Causes Sterile Inflammation

    doi: 10.3389/fimmu.2018.00705

    Figure Lengend Snippet: High-mobility group box 1 (HMGB1) B box activates complement cascade. (A) Schematic overview of recombinant HMGB1 proteins used: wild-type (WT) HMGB1, boxes A (aa 1-79) and B (aa 88-162), ΔC-HMGB1 (aa 1-185), and ΔN-HMGB1 (aa 11-215). (B,C) Complement consumption assessment. WT HMGB1 proteins (B) or HMGB1 variants (C) were incubated with the diluted normal human serum (NHS) containing CH 50 activity in GVB 2+ buffer for 30 min at 37°C. After complement consumption by HMGB1, sRBCs were added in the consumed NHS for 30 min at 37°C. Aggregated human IgG and IgG-free BSA (each 30 µg/ml) were used as the positive and negative controls, respectively. Data shown are the mean ± SD of three independent repeats. * P

    Article Snippet: Rabbit anti-HMGB1 Ab (1:1,500, Abcam #18256) was added for 1 h at RT after washing.

    Techniques: Recombinant, Incubation, Activity Assay

    High-mobility group box 1 (HMGB1)-mediated membrane attack complex (MAC) formation and its effect to cell signaling. (A,B) MEFs were cultured in DMEM containing 10% normal human serum (NHS) in the presence or absence of 1 µg/ml HMGB1 for 1 h at 37°C. Sublytic MAC proteins were stained using anti-C5b-9 antibody (Ab) (red) and observed by confocal microscopy. Blue: DAPI. Heat-inactivated (HI) NHS was used. Scale bar, 10 µm (A) . MEFs were incubated with 10% NHS in the presence of different concentrations of HMGB1, and then the mean relative intensity of fluorescence of 10 visual fields was calculated (B) . Error bars are mean ± SD. * P

    Journal: Frontiers in Immunology

    Article Title: High-Mobility Group Box 1-Induced Complement Activation Causes Sterile Inflammation

    doi: 10.3389/fimmu.2018.00705

    Figure Lengend Snippet: High-mobility group box 1 (HMGB1)-mediated membrane attack complex (MAC) formation and its effect to cell signaling. (A,B) MEFs were cultured in DMEM containing 10% normal human serum (NHS) in the presence or absence of 1 µg/ml HMGB1 for 1 h at 37°C. Sublytic MAC proteins were stained using anti-C5b-9 antibody (Ab) (red) and observed by confocal microscopy. Blue: DAPI. Heat-inactivated (HI) NHS was used. Scale bar, 10 µm (A) . MEFs were incubated with 10% NHS in the presence of different concentrations of HMGB1, and then the mean relative intensity of fluorescence of 10 visual fields was calculated (B) . Error bars are mean ± SD. * P

    Article Snippet: Rabbit anti-HMGB1 Ab (1:1,500, Abcam #18256) was added for 1 h at RT after washing.

    Techniques: Cell Culture, Staining, Confocal Microscopy, Incubation, Fluorescence

    High-mobility group box 1 (HMGB1)-induced complement activation is decreased in C1q-deficient mice of APAP-induced hepatotoxicity model. (A) Overnight fasted wild-type (WT) or C1q −/− mice (9–10 weeks old, male) were intraperitoneally (i.p.) treated with 400 mg/kg of APAP or saline. After 0, 6, and 24 h, mouse serum samples were collected. Serum alanine aminotransferase (ALT) activity (left) and C3 levels (right) were determined using ALT color endpoint assay and enzyme-linked immunosorbent assay, respectively. Each dot represents each mouse with triplicates per sample (four or five mice per group). (B,C) Mice were euthanized 24 h after APAP administration, and the left medial lobe of liver was fixed in 4% paraformaldehyde and 30% sucrose. Liver tissue was stained with hematoxylin and eosin for evaluation of necrosis and hemorrhage (B) . Tissue-Tec OCT-embedded liver was stained with anti-C1q and anti-HMGB1 antibodies (Abs) for immunofluorescence analysis (C) . (D) Serum HMGB1 protein from WT and C1q −/− mice was detected using Western blot analysis 24 h after APAP injection. (E) Anti-HMGB1 (2G7) or mouse IgG (5 μg/mouse) was i.p. administrated immediately after APAP administration to block HMGB1. After 24 h, mouse serum samples were collected and ALT activity (left panel) and concentration of C3 (right panel) were determined. Saline was used as a negative control. (F) HMGB1 was i.p. injected with sRAGE (5 µg/mouse) as described in (E) . N = 4–5 mice/group. Data = mean ± SEM. Student’s t -test (unpaired two-tailed) was used to calculate the P -value. All scale bars, 20 µm. Data are representative of three (A–E) or two (F) independent experiments.

    Journal: Frontiers in Immunology

    Article Title: High-Mobility Group Box 1-Induced Complement Activation Causes Sterile Inflammation

    doi: 10.3389/fimmu.2018.00705

    Figure Lengend Snippet: High-mobility group box 1 (HMGB1)-induced complement activation is decreased in C1q-deficient mice of APAP-induced hepatotoxicity model. (A) Overnight fasted wild-type (WT) or C1q −/− mice (9–10 weeks old, male) were intraperitoneally (i.p.) treated with 400 mg/kg of APAP or saline. After 0, 6, and 24 h, mouse serum samples were collected. Serum alanine aminotransferase (ALT) activity (left) and C3 levels (right) were determined using ALT color endpoint assay and enzyme-linked immunosorbent assay, respectively. Each dot represents each mouse with triplicates per sample (four or five mice per group). (B,C) Mice were euthanized 24 h after APAP administration, and the left medial lobe of liver was fixed in 4% paraformaldehyde and 30% sucrose. Liver tissue was stained with hematoxylin and eosin for evaluation of necrosis and hemorrhage (B) . Tissue-Tec OCT-embedded liver was stained with anti-C1q and anti-HMGB1 antibodies (Abs) for immunofluorescence analysis (C) . (D) Serum HMGB1 protein from WT and C1q −/− mice was detected using Western blot analysis 24 h after APAP injection. (E) Anti-HMGB1 (2G7) or mouse IgG (5 μg/mouse) was i.p. administrated immediately after APAP administration to block HMGB1. After 24 h, mouse serum samples were collected and ALT activity (left panel) and concentration of C3 (right panel) were determined. Saline was used as a negative control. (F) HMGB1 was i.p. injected with sRAGE (5 µg/mouse) as described in (E) . N = 4–5 mice/group. Data = mean ± SEM. Student’s t -test (unpaired two-tailed) was used to calculate the P -value. All scale bars, 20 µm. Data are representative of three (A–E) or two (F) independent experiments.

    Article Snippet: Rabbit anti-HMGB1 Ab (1:1,500, Abcam #18256) was added for 1 h at RT after washing.

    Techniques: Activation Assay, Mouse Assay, Activity Assay, End Point Assay, Enzyme-linked Immunosorbent Assay, Staining, Immunofluorescence, Western Blot, Injection, Blocking Assay, Concentration Assay, Negative Control, Two Tailed Test

    Binding of high-mobility group box 1 (HMGB1) to C1q. (A) Purified C1q protein (10 µg/ml) was immobilized on a microtiter plate, and different concentrations of HMGB1 protein were added for an enzyme-linked immunosorbent assay (ELISA). Buffer was used as a negative control. Data are representative of three independent experiments. Error bars are mean ± SD. * P

    Journal: Frontiers in Immunology

    Article Title: High-Mobility Group Box 1-Induced Complement Activation Causes Sterile Inflammation

    doi: 10.3389/fimmu.2018.00705

    Figure Lengend Snippet: Binding of high-mobility group box 1 (HMGB1) to C1q. (A) Purified C1q protein (10 µg/ml) was immobilized on a microtiter plate, and different concentrations of HMGB1 protein were added for an enzyme-linked immunosorbent assay (ELISA). Buffer was used as a negative control. Data are representative of three independent experiments. Error bars are mean ± SD. * P

    Article Snippet: Rabbit anti-HMGB1 Ab (1:1,500, Abcam #18256) was added for 1 h at RT after washing.

    Techniques: Binding Assay, Purification, Enzyme-linked Immunosorbent Assay, Negative Control

    Cardiac cells express the HMGB1 receptors: RAGE and TLR4 receptor-4. These expressions were evaluated at the mRNA level [reverse transcriptase–polymerase chain reaction ( A )] and at the protein level [western immunoblotting ( B )]

    Journal: Cardiovascular Research

    Article Title: Peroxynitrite induces HMGB1 release by cardiac cells in vitro and HMGB1 upregulation in the infarcted myocardium in vivo

    doi: 10.1093/cvr/cvq373

    Figure Lengend Snippet: Cardiac cells express the HMGB1 receptors: RAGE and TLR4 receptor-4. These expressions were evaluated at the mRNA level [reverse transcriptase–polymerase chain reaction ( A )] and at the protein level [western immunoblotting ( B )]

    Article Snippet: At the end of the reperfusion period, hearts were removed for the determination of myocardial infarct size (2 h reperfusion) and for the evaluation of the expression levels of HMGB1 and nitrotyrosine (1 h reperfusion) within the cardiac tissue (discussed subsequently).

    Techniques: Polymerase Chain Reaction, Western Blot

    Necrotic cells, but not apoptotic, released HMGB1 upon peroxynitrite stimulation. Cardiac non-myocyte cells treated with 100 μM peroxynitrite for 40 min followed by 4 h in the culture medium were stained with propidium iodide and Cy™5

    Journal: Cardiovascular Research

    Article Title: Peroxynitrite induces HMGB1 release by cardiac cells in vitro and HMGB1 upregulation in the infarcted myocardium in vivo

    doi: 10.1093/cvr/cvq373

    Figure Lengend Snippet: Necrotic cells, but not apoptotic, released HMGB1 upon peroxynitrite stimulation. Cardiac non-myocyte cells treated with 100 μM peroxynitrite for 40 min followed by 4 h in the culture medium were stained with propidium iodide and Cy™5

    Article Snippet: At the end of the reperfusion period, hearts were removed for the determination of myocardial infarct size (2 h reperfusion) and for the evaluation of the expression levels of HMGB1 and nitrotyrosine (1 h reperfusion) within the cardiac tissue (discussed subsequently).

    Techniques: Staining

    Necrosis and passive release of HMGB1 are induced by peroxynitrite in primary murine cardiomyocytes and non-myocyte cells. Neonatal murine cardiomyocytes (left panel) and non-myocyte cells (middle panel) exposed to 100 μM peroxynitrite for 40

    Journal: Cardiovascular Research

    Article Title: Peroxynitrite induces HMGB1 release by cardiac cells in vitro and HMGB1 upregulation in the infarcted myocardium in vivo

    doi: 10.1093/cvr/cvq373

    Figure Lengend Snippet: Necrosis and passive release of HMGB1 are induced by peroxynitrite in primary murine cardiomyocytes and non-myocyte cells. Neonatal murine cardiomyocytes (left panel) and non-myocyte cells (middle panel) exposed to 100 μM peroxynitrite for 40

    Article Snippet: At the end of the reperfusion period, hearts were removed for the determination of myocardial infarct size (2 h reperfusion) and for the evaluation of the expression levels of HMGB1 and nitrotyrosine (1 h reperfusion) within the cardiac tissue (discussed subsequently).

    Techniques:

    Peroxynitrite induces necrosis of H9c2 cardiomyoblasts and the passive release of HMGB1. H9c2 cells were exposed to 100–250 μM peroxynitrite for 40 min and then replaced in the culture medium for a total of 4 h. Cell necrosis was evaluated

    Journal: Cardiovascular Research

    Article Title: Peroxynitrite induces HMGB1 release by cardiac cells in vitro and HMGB1 upregulation in the infarcted myocardium in vivo

    doi: 10.1093/cvr/cvq373

    Figure Lengend Snippet: Peroxynitrite induces necrosis of H9c2 cardiomyoblasts and the passive release of HMGB1. H9c2 cells were exposed to 100–250 μM peroxynitrite for 40 min and then replaced in the culture medium for a total of 4 h. Cell necrosis was evaluated

    Article Snippet: At the end of the reperfusion period, hearts were removed for the determination of myocardial infarct size (2 h reperfusion) and for the evaluation of the expression levels of HMGB1 and nitrotyrosine (1 h reperfusion) within the cardiac tissue (discussed subsequently).

    Techniques:

    Peroxynitrite decomposition catalysts reduce myocardial necrosis and suppress myocardial HMGB1 overexpression induced by ischaemia–reperfusion. Rats challenged with 45 min myocardial ischaemia followed by reperfusion (1–2 h) were left

    Journal: Cardiovascular Research

    Article Title: Peroxynitrite induces HMGB1 release by cardiac cells in vitro and HMGB1 upregulation in the infarcted myocardium in vivo

    doi: 10.1093/cvr/cvq373

    Figure Lengend Snippet: Peroxynitrite decomposition catalysts reduce myocardial necrosis and suppress myocardial HMGB1 overexpression induced by ischaemia–reperfusion. Rats challenged with 45 min myocardial ischaemia followed by reperfusion (1–2 h) were left

    Article Snippet: At the end of the reperfusion period, hearts were removed for the determination of myocardial infarct size (2 h reperfusion) and for the evaluation of the expression levels of HMGB1 and nitrotyrosine (1 h reperfusion) within the cardiac tissue (discussed subsequently).

    Techniques: Over Expression