Journal: Kidney International

Article Title: Early interleukin 6 production by leukocytes during ischemic acute kidney injury is regulated by TLR4

doi: 10.1038/ki.2011.140

Figure Lengend Snippet: Interleukin 6 (IL6) was measured by quantitative reverse transcriptase-PCR. Each sample normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The calibrator gene is the media-treated BMDM. n = 4. Means and standard deviation are shown. *P<0.05 between TLR4 (+/+) media, HSP70, and HMGB1 groups. TLR4 (−/−) mice were C57BL/10ScNJ, and TLR4 (+/+) mice were C57BL/10SnJ.

Article Snippet: Macrophage cells were treated with either ROS-stressed S3 supernatant, recombinant human HMGB1 (rhHMGB1, R&D Systems) with or without 100 μmol/l glycyrrhizic acid (Sigma), or heat shock protein 70 (Stressgen, San Diego, CA) for 4 h. Treatment of S3 Cells with ROS The SV-40 transformed S3 tubular cells 83 were maintained as previously described.

Techniques: Reverse Transcription, Standard Deviation

Journal: Kidney International

Article Title: Early interleukin 6 production by leukocytes during ischemic acute kidney injury is regulated by TLR4

doi: 10.1038/ki.2011.140

Figure Lengend Snippet: (a) Experimental design. In stage I, the SV40-transformed S3 tubules were a gift from Dr Glenn Nagami.83 If they were injured by ROS, they released endogenous TLR4 ligands into their supernatant for 12 h. In stage II, the supernatant was cultured with murine bone marrow macrophages. (b) Only TLR4 (+/+) macrophages are activated by supernatant from ROS-stimulated S3 cells. TLR4 (+/+) macrophages are from C57BL/10SnJ mice. TLR4 (−/−) macrophages are from C57BL/10ScNJ mice. Results of typical experiment from four are shown. n = 3, mean and standard error are shown. *P<0.05 between S3 + ROS/TLR4 (+/+) macrophage and other groups by t-test. (c) High-mobility group protein B1 (HMGB1) in supernatants. n = 4. Mean and standard errors shown. P<0.05 between no treatment and ROS groups. IL6, interleukin 6.

Article Snippet: Macrophage cells were treated with either ROS-stressed S3 supernatant, recombinant human HMGB1 (rhHMGB1, R&D Systems) with or without 100 μmol/l glycyrrhizic acid (Sigma), or heat shock protein 70 (Stressgen, San Diego, CA) for 4 h. Treatment of S3 Cells with ROS The SV-40 transformed S3 tubular cells 83 were maintained as previously described.

Techniques: Transformation Assay, Cell Culture

Journal: Oncology reports

Article Title: Invasion potential of H22 hepatocarcinoma cells is increased by HMGB1-induced tumor NF-κB signaling via initiation of HSP70.

doi: 10.3892/or.2013.2595

Figure Lengend Snippet: Figure 1. Extracellular high-mobility group protein B1 (HMGB1) is increased in the tumor microenvironment during tumor progression by DTC-Ms and heat shock protein (HSP) 70. (A and B) Effect of DTC-Ms and HSP70 on tumor growth. (A) BALB/c mice were inoculated with H22 cells on day (d) 0 and received intramuscular injection of DTC-Ms and HSP70 (200 µg/mouse), once every 2 days from d1 to 13. Tumors (n=8 in each group) were dissected and weighed on d15 (d1-13 injection) after tumor inoculation. *P<0.05. (B) Detection of HMGB1 in tumors. Extracellular HMGB1 was detected by western blot analysis at the indicated time point after inoculation as described in Materials and methods. Tissues adjacent to the tumor were used as controls. (C) Expression of sTLR2 and sTLR4 after intramuscular transfection. Naked plasmid DNA was injected into muscle of mice. Tissues at sites of injection were surgically excised 72 h later and homogenized. The expressions of sTLR2 and sTLR4 were identified by reverse transcriptase-PCR and western blot analysis. (D) Flow cytometric analysis of TLR2 and TLR4 expression on H22 cells. (E and F) sTLR2 and sTLR4 suppressed HSP70 production but not HMGB1 production. (E) Mice inoculated with H22 cells received intramuscular injection of saline, pcDNA3.1, or psTLR2/psTLR4, with simultaneous injection of HSP70 as described in Materials and methods. Tumors (n=8 in each group) were dissected and weighed on d15 after tumor inoculation. *P<0.05. (F) Extracellular HMGB1 was detected by western blot analysis at the indicated time point after inoculation as described in Materials and methods.

Article Snippet: HMGB1 antibody was purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Injection, Western Blot, Expressing, Transfection, Plasmid Preparation, Reverse Transcription, Saline

Journal: Oncology reports

Article Title: Invasion potential of H22 hepatocarcinoma cells is increased by HMGB1-induced tumor NF-κB signaling via initiation of HSP70.

doi: 10.3892/or.2013.2595

Figure Lengend Snippet: Figure 2. Tumor cells exhibit an increased invasive growth by heat shock protein (HSP) 70‑induced expression of high-mobility group protein B1 (HMGB1). (A and B) H22 cells or HMGB1shRNA expressing H22 cells were treated in vitro and then inoculated to the liver of mice. Mice received no treatment or were administered HSP70 or HMGB1 as described in Materials and methods. (A) The invasive growth of tumor cells after HSP70 or HMGB1 treatment. The tumors in the liver of the mice are shown (upper). The main tumor nodules were measured (lower, left) and satellite tumor nodes counted (lower, right). *P<0.05, compared with control or saline groups. No satellite nodes were observed in control or the HMGB1shRNA mice administered H22 tumors. (B) On day (d) 16 after inoculation, tumor marginal tissues were surgically removed to extract RNA and proteins. The relative mRNA levels of MMP‑9 were detected by real‑time reverse transcriptase (RT)‑PCR (left). *P<0.05, compared with control groups. MMP‑9 in supernatants was detected by zymography assay (right). (C) Assay of MMP‑9 production. Cells were cultured in the presence or absence of HSP70 or HMGB1 for 5 or 24 h. The relative mRNA levels of MMP‑9 were detected by real‑time RT‑PCR (left). *P<0.05, compared with control groups. MMP‑9 in supernatants was detected by zymography assay (right).

Article Snippet: HMGB1 antibody was purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, In Vitro, Control, Saline, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Zymography, Cell Culture, Quantitative RT-PCR

Journal: Oncology reports

Article Title: Invasion potential of H22 hepatocarcinoma cells is increased by HMGB1-induced tumor NF-κB signaling via initiation of HSP70.

doi: 10.3892/or.2013.2595

Figure Lengend Snippet: Figure 5. Receptor for advanced glycation end products (RAGE) but not Toll-like receptor (TLR)2/TLR4 is required for high-mobility group protein B1 (HMGB1)‑mediated MMP‑9 enhancement. (A) H22 cells were transfected with the indicated RAGE‑shRNA for 48 h and were then cultured in the presence of HMGB1 and/or resveratrol (Res, 30 µM). MMP‑9 in supernatants was detected by zymography assay. (B) H22 cells were cultured in the presence of HMGB1 and QNZ (20 nM) or Res (30 µM), and NF‑κB phosphorylation was analyzed by western blotting. (C) HepG2 cells were transfected with the indicated RAGE‑shRNA for 48 h and were then cultured in the presence of HMGB1 and/or Res (30 µM). MMP‑9 in supernatants was detected by zymography assay.

Article Snippet: HMGB1 antibody was purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Transfection, Cell Culture, Zymography, Phospho-proteomics, Western Blot

Journal: Oncology reports

Article Title: Invasion potential of H22 hepatocarcinoma cells is increased by HMGB1-induced tumor NF-κB signaling via initiation of HSP70.

doi: 10.3892/or.2013.2595

Figure Lengend Snippet: Figure 4. Time course analysis of heat shock protein (HSP) 70‑induced high-mobility group protein B1 (HMGB1) and Beclin‑1 production and effect of Jun N-terminal kinase (JNK) inhibitors on HSP70‑induced NF‑κB activation. (A) Effect of HSP70 on production of HMGB1 and Beclin‑1. H22 cells were incubated with HSP70 (20 µg/ml) for various times and Beclin‑1 and HMGB1 protein production was analyzed over a 6‑h period. Supernatants were collected at various times after HSP70 stimulation and the concentration of Beclin‑1 and HMGB1 was determined by western blotting and compared with basal levels (time 0). (B) A JNK specific inhibitor inhibited JNK activation. Cells were treated with and/or without JNK specific inhibitor (SP600125, 10 µmol/l) followed by stimulation with HSP70 (20 µg/ml) for 4 h. JNK and p‑JNKs were measured by western blotting. (C) Activation of JNK is required for HSP70‑induced Beclin‑1 and HMGB1 production. Cells were treated with and/or without a JNK specific inhibitor (SP600125, 10 µmol/l) and/or Beclin‑1 shRNA followed by stimulation with HSP70 (20 µg/ml) for 4 h. Empty vector served as a negative control. HMGB1 and Beclin‑1 were measured by western blotting. (D) Effects on NF‑κB activation in H22 cells. H22 cells were incubated with HMGB1 (20 µg/ml) or with a JNK specific inhibitor (SP600125, 10 µmol/l) followed by stimulation with HSP70 (20 µg/ml) for various times (0‑8 h), and NF‑κB phosphorylation was analyzed by western blotting.

Article Snippet: HMGB1 antibody was purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Activation Assay, Incubation, Concentration Assay, Western Blot, shRNA, Plasmid Preparation, Negative Control, Phospho-proteomics

Journal: PLoS ONE

Article Title: Expression of RAGE and HMGB1 in Thymic Epithelial Tumors, Thymic Hyperplasia and Regular Thymic Morphology

doi: 10.1371/journal.pone.0094118

Figure Lengend Snippet: Analysis of immunohistochemical staining of RAGE and HMGB1 for TETs.

Article Snippet: Sections were stained using affinity-purified polyclonal goat anti-human RAGE IgG (R&D Systems, Minneapolis, MN, USA) or monoclonal mouse anti-human HMGB1 IgG 2b (R&D Systems) and biotinylated anti-goat IgG or anti-mouse IgG secondary antibodies (Vector Laboratories, Burlingame, CA, USA).

Techniques: Immunohistochemical staining, Staining

Journal: PLoS ONE

Article Title: Expression of RAGE and HMGB1 in Thymic Epithelial Tumors, Thymic Hyperplasia and Regular Thymic Morphology

doi: 10.1371/journal.pone.0094118

Figure Lengend Snippet: Expression of HMGB1 in thymoma type A (A), B-component type AB (B), B1 (C) is shown. Scale bar: 40 μm. On this example of B1 thymoma lymphocytes are intermingled with few tumor cells. Focus on cytoplasmic staining: a larger magnification of a WHO type B1 thymoma is shown to better display “autophagic” tumor cells (tumor cells with brownish granular cytoplasmic and absent [only hematoxylin blue] nuclear staining). Scale bar: 20 μm (D). Analogously, a type B2 (E) and B3 (F) thymoma are shown. Two examples of TC (SCC (G) and (H)) are displayed – scale bar: 40 μm. HMGB1 high mobility group box1, TETs thymic epithelial tumors, TC thymic carcinoma, SCC squamous cell carcinoma

Article Snippet: Sections were stained using affinity-purified polyclonal goat anti-human RAGE IgG (R&D Systems, Minneapolis, MN, USA) or monoclonal mouse anti-human HMGB1 IgG 2b (R&D Systems) and biotinylated anti-goat IgG or anti-mouse IgG secondary antibodies (Vector Laboratories, Burlingame, CA, USA).

Techniques: Expressing, Staining

Journal: PLoS ONE

Article Title: Expression of RAGE and HMGB1 in Thymic Epithelial Tumors, Thymic Hyperplasia and Regular Thymic Morphology

doi: 10.1371/journal.pone.0094118

Figure Lengend Snippet: Immunohistochemistry revealed strong expression of RAGE in subcapsular cTEC of fetal (A) and adult thymus (B). Scale bar: 40 μm. For comparison the staining pattern of cytokeratins 5 and 14 – markers of epithelial cell origin on fetal thymus is shown (C). The expression pattern for HMGB1 in cTEC of fetal (D, scale bar: 40 μm) and adult thymus (E, scale bar: 20 μm) is shown. (F) A larger magnification of E is shown. Scale bar 8 μm. Arrows in F indicate HMGB1 cytoplasmic staining in cTEC. RAGE receptor for advanced glycation endproducts, HMGB1 high mobility group box1, cTEC cortical thymic epithelial cells

Article Snippet: Sections were stained using affinity-purified polyclonal goat anti-human RAGE IgG (R&D Systems, Minneapolis, MN, USA) or monoclonal mouse anti-human HMGB1 IgG 2b (R&D Systems) and biotinylated anti-goat IgG or anti-mouse IgG secondary antibodies (Vector Laboratories, Burlingame, CA, USA).

Techniques: Immunohistochemistry, Expressing, Comparison, Staining

Journal: PLoS ONE

Article Title: Expression of RAGE and HMGB1 in Thymic Epithelial Tumors, Thymic Hyperplasia and Regular Thymic Morphology

doi: 10.1371/journal.pone.0094118

Figure Lengend Snippet: Hassall's corpuscles stained with antibodies to RAGE (A), and HMGB1 (B, small arrows point to small GC) are displayed. Scale bar: 80 μm. Similarly, RAGE (C), and HMGB1 (D) staining in GC of MG patients (scale bar: 80 μm); and RAGE expression in thymic medulla (E, arrows point to thymic medulla; scale bar: 200 μm) and macrophages (F, scale bar: 40 μm) of regular adult thymus are shown. RAGE receptor for advanced glycation endproducts, HMGB1 high mobility group box1, MG Myasthenia gravis, GC germinal center.

Article Snippet: Sections were stained using affinity-purified polyclonal goat anti-human RAGE IgG (R&D Systems, Minneapolis, MN, USA) or monoclonal mouse anti-human HMGB1 IgG 2b (R&D Systems) and biotinylated anti-goat IgG or anti-mouse IgG secondary antibodies (Vector Laboratories, Burlingame, CA, USA).

Techniques: Staining, Expressing

Journal: PLoS ONE

Article Title: Expression of RAGE and HMGB1 in Thymic Epithelial Tumors, Thymic Hyperplasia and Regular Thymic Morphology

doi: 10.1371/journal.pone.0094118

Figure Lengend Snippet: Concentration of sRAGE, esRAGE and HMGB1 in serum of patients.

Article Snippet: Sections were stained using affinity-purified polyclonal goat anti-human RAGE IgG (R&D Systems, Minneapolis, MN, USA) or monoclonal mouse anti-human HMGB1 IgG 2b (R&D Systems) and biotinylated anti-goat IgG or anti-mouse IgG secondary antibodies (Vector Laboratories, Burlingame, CA, USA).

Techniques: Concentration Assay

Journal: PLoS ONE

Article Title: Expression of RAGE and HMGB1 in Thymic Epithelial Tumors, Thymic Hyperplasia and Regular Thymic Morphology

doi: 10.1371/journal.pone.0094118

Figure Lengend Snippet: Levels of sRAGE (a), esRAGE (b) and HMGB1 (c) in sera of patients with TETs including patients with paraneoplastic MG (MG n = 11) compared to healthy volunteers are shown. To rule out the influence of MG on levels of circulating sRAGE (d) and HMGB1 (e) in patients with TETs, patients with MG were excluded from this analysis. The levels of sRAGE in non-invasive (Masaoka-Koga stage I) and invasive TETs (Masaoka-Koga stages II-IV) are shown (f). RAGE receptor for advanced glycation endproducts, sRAGE soluble RAGE, esRAGE endogenous secretory RAGE, HMGB1 high mobility group box1, TETs thymic epithelial tumors, n number, MG Myasthenia gravis.

Article Snippet: Sections were stained using affinity-purified polyclonal goat anti-human RAGE IgG (R&D Systems, Minneapolis, MN, USA) or monoclonal mouse anti-human HMGB1 IgG 2b (R&D Systems) and biotinylated anti-goat IgG or anti-mouse IgG secondary antibodies (Vector Laboratories, Burlingame, CA, USA).

Techniques:

Journal: PLoS ONE

Article Title: Expression of RAGE and HMGB1 in Thymic Epithelial Tumors, Thymic Hyperplasia and Regular Thymic Morphology

doi: 10.1371/journal.pone.0094118

Figure Lengend Snippet: The levels of circulating sRAGE (a), and HMGB1 (b) in serum of patients with TETs compared to patients with thymic hyperplasia and healthy volunteers are shown. RAGE receptor for advanced glycation endproducts, sRAGE soluble RAGE, HMGB1 high mobility group box1, TET thymic epithelial tumor.

Article Snippet: Sections were stained using affinity-purified polyclonal goat anti-human RAGE IgG (R&D Systems, Minneapolis, MN, USA) or monoclonal mouse anti-human HMGB1 IgG 2b (R&D Systems) and biotinylated anti-goat IgG or anti-mouse IgG secondary antibodies (Vector Laboratories, Burlingame, CA, USA).

Techniques:

Journal: PLoS ONE

Article Title: Expression of RAGE and HMGB1 in Thymic Epithelial Tumors, Thymic Hyperplasia and Regular Thymic Morphology

doi: 10.1371/journal.pone.0094118

Figure Lengend Snippet: Patients with TETs were separated into patients with thymomas and TC, and compared to healthy volunteers. Serum concentrations of sRAGE (a) and HMGB1 (b) are shown. Patients with TC were analyzed compared to volunteers for sRAGE (c), esRAGE (d), as well as HMGB1 (e). TETs thymic epithelial tumors, RAGE receptor for advanced glycation endproducts, sRAGE soluble RAGE, esRAGE endogenous secretory RAGE, HMGB1 high mobility group box1, TC thymic carcinoma

Article Snippet: Sections were stained using affinity-purified polyclonal goat anti-human RAGE IgG (R&D Systems, Minneapolis, MN, USA) or monoclonal mouse anti-human HMGB1 IgG 2b (R&D Systems) and biotinylated anti-goat IgG or anti-mouse IgG secondary antibodies (Vector Laboratories, Burlingame, CA, USA).

Techniques: