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Hmgb1

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HMGB1 is a nuclear protein that functions as a DNA-binding and DNA-bending protein. It plays a role in the regulation of gene expression and is involved in various cellular processes.

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Lab products found in correlation

Bradford bca colorimetric assay, by Bio-Rad (2 mentions) Cox 2, by Cell Signaling Technology (2 mentions) Gapdh, by Santa Cruz Biotechnology (2 mentions) Heat inactivated fbs, by Thermo Fisher Scientific (1 mentions) Nci h460, by American Type Culture Collection (1 mentions) Tnf α, by Cell Signaling Technology (1 mentions) Il 1β, by Cell Signaling Technology (1 mentions) Ultra multifunctional microplate reader, by Tecan (1 mentions) Linoleic acid, by Merck Group (1 mentions) Cellstripper, by Corning (1 mentions) Chloroquine, by Fujifilm (1 mentions) Ferrostatin 1, by Cayman Chemical (1 mentions) N acetyl l cysteine nac, by Merck Group (1 mentions) Z vad fmk zvad, by Santa Cruz Biotechnology (1 mentions) Deferoxamine dfo, by Cayman Chemical (1 mentions) Bay11 7082, by Merck Group (1 mentions) Imd 0354, by Merck Group (1 mentions) Tnf α, by Merck Group (1 mentions) Recombinant human tnf α, by Thermo Fisher Scientific (1 mentions) Hmgb1, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

Anti-HMGB1 Mouse anti-HMGB1 HMGB1 antibody Recombinant human HMGB1 HMGB1 protein

16 protocols using hmgb1

1

Cell Line Maintenance and Drug Sourcing

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Cell lines (i.e., NCI-H460, A549) were purchased from American type culture collection (ATCC, USA). Cell lines were grown and maintained using RPMI-1640 (Hyclone, USA) supplemented with 10% heat inactivated-FBS (GIBCO, Auckland, NZ) in an incubation system at 37 °C with 5% CO2. DOC was obtained from Shanghai Sunve Pharmaceutical Co. Ltd. L-OHP was obtained from Shenzhen Sea King biological engineering Limited by Share Ltd. Recombinant protein HMGB1was purchased from BioLegend (San Diego, CA, USA).
Gao Q., Wang S., Chen X., Cheng S., Zhang Z., Li F., Huang L., Yang Y., Zhou B., Yue D., Wang D., Cao L., Maimela N.R., Zhang B., Yu J., Wang L, & Zhang Y. (2019). Cancer-cell-secreted CXCL11 promoted CD8+ T cells infiltration through docetaxel-induced-release of HMGB1 in NSCLC. Journal for Immunotherapy of Cancer, 7, 42.
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2

Chondrogenic Cell Viability Assay

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Cell viability of chondrogenic C3H10T1/2 cells was assessed using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT, M2128, Sigma-Aldrich, St. Louis, MO, USA) assay. Briefly, chondrogenic C3H10T1/2 cells were plated at a density of 1 × 104 cells/well in 24-well culture plates and incubated for 24 h at 37 °C and 5% CO2. The next day, the culture medium was replaced with fresh Dulbecco’s modified Eagle’s medium (DMEM; vehicle control) or DMEM containing different concentrations of reagents: H2O2 (Sigma-Aldrich), LPS (E. coli 026:B6, L2654, Sigma-Aldrich), IL-1β (Cell Signaling Technology, Danvers, MA, USA), IL-6 (CYT-213, PROSPEC, Israel), TNFα (Cell Signaling Technology), and HMGB1 (BioLegend Inc., San Diego, CA, USA). At 48 h after reagent treatment, MTT (5 mg/mL) was added, and the culture plates were incubated at 37 °C for 4 h. The resulting formazan product was dissolved in DMSO, and the absorbance was measured at 560 nm using an Ultra Multifunctional Microplate Reader (Tecan US, Inc., Morrisville, NC, USA). Cell viability was determined from the readings using the formula % Viability = (fluorescence value of MSM/fluorescence value of non-treated control) × 100. All measurements were performed in triplicate, and the experiments were repeated at least thrice.
Kyung B.S., Jung K.W., Yeo W.J., Seo H.K., Lee Y.S, & Suh D.W. (2021). Differential regulation of the water channel protein aquaporins in chondrocytes of human knee articular cartilage by aging. Scientific Reports, 11, 20425.
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3

Macrophage Response to Exosomes and TLR Ligands

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Peritoneal macrophages were treated with MLE-12 Exo or LLC exo (40μg/mL) for 16 hours. After incubation, supernatant was removed and cells were washed briefly with PBS. Cells were harvested by adding 500μL Cell Stripper (Corning) and incubated for 1 minute at 37°C. RPMI-1640 was added to quench the reaction and cells were gently removed by scraping. For NF-κB experiments, macrophages were pre-treated with 0.2 μg/mL BAY-11-7082 for 1 hour at 37°C. Cells were then washed with PBS and then treated with exosome stimulation as normal. For the HMGB-1 studies, cells were stimulated with 10μg/mL recombinant murine HMGB-1 (Biolegend) for 16 hours. Macrophages were stimulated with 25μM linoleic acid (Sigma) for 16 hours. Peritoneal macrophages were sorted from C57BL/6 WT, TLR2−/− and TLR7−/− mice. 1×105 macrophages were plated and were stimulated with 4T1 TDE (5 μg/ml), exosomal RNA (140ng/mL), LPS (10 ng/ml), CpG ODN (1 μg/ml), and CL075 (1 μg/ml) for 16 hours. Culture supernatants were collected and assayed for TNF-α and IL-6 by ELISA.
Morrissey S.M., Zhang F., Ding C., Montoya-Durango D.E., Hu X., Yang C., Wang Z., Yuan F., Fox M., Zhang H.G., Guo H., Tieri D., Kong M., Watson C.T., Mitchell R.A., Zhang X., McMasters K.M., Huang J, & Yan J. (2021). Tumor-derived Exosomes Drive Immunosuppressive Macrophages in a Pre-metastatic Niche through Glycolytic Dominant Metabolic Reprogramming. Cell metabolism, 33(10), 2040-2058.e10.
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4

Cell Death Inhibitor Assay in H9c2 Cells

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For the cell death inhibitor assay, H9c2 cells were treated with human recombinant HMGB1 (40 μg/mL, Biolegend, San Diego, CA, USA) for 48 h, with or without the addition of cell death inhibitors. The following inhibitors were utilized: N-acetyl-L-cysteine (NAC, 1 mM, Sigma, St. Louis, MO, USA), Z-VAD-FMK (ZVAD, 20 μM, Santa Cruz Biotechnology, Santa Cruz, CA, USA), ferrostatin-1 (FRS, 2 μM), deferoxamine (DFO, 200 μM, Cayman Chemicals, Ann Arbor, MI, USA), and 4-phenylbutyric acid (4PBA, 200 μM, WAKO). Additionally, chloroquine (100 µM, WAKO) was employed to inhibit autophagy.
Goto K., Fujiwara-Tani R., Nukaga S., Miyagawa Y., Kawahara I., Nishida R., Ikemoto A., Sasaki R., Ogata R., Kishi S., Luo Y., Fujii K., Ohmori H, & Kuniyasu H. (2024). Berberine Improves Cancer-Derived Myocardial Impairment in Experimental Cachexia Models by Targeting High-Mobility Group Box-1. International Journal of Molecular Sciences, 25(9), 4735.
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5

NRVM Supernatant and HMGB1 Effects on Cardiac Fibroblasts

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CFs were isolated and cultured, as described previously (Li F. et al., 2018 (link)). The CFs passaged to the 2–4th generation were used for the experiment. Cells were seeded in 6- or 24-well plates and stimulated with ISO (10 μM) or PBS to determine the effect of NRVMs supernatant on the activation and proliferation of CFs. We cultured CFs in the supernatant of NRVMs treated with ISO and siNLRP3 or Ad-NLRP3 for 24 h. Different concentrations (0, 2, 4, 8, and 16 ng/ml) of recombinant human high-mobility group box 1 protein (HMGB1; Biolegend, San Diego, United States) were used to treat CFs in the cell proliferation experiment. The concentration of 16 ng/ml HMGB1 was used for further study, and glycyrrhizic acid (MCE, New Jersey, United States) was used to antagonize HMGB1.
Liu C., Hu T., Cai Z., Xie Q., Yuan Y., Li N., Xie S., Yao Q., Zhao J., Wu Q.Q, & Tang Q. (2020). Nucleotide-Binding Oligomerization Domain-Like Receptor 3 Deficiency Attenuated Isoproterenol-Induced Cardiac Fibrosis via Reactive Oxygen Species/High Mobility Group Box 1 Protein Axis. Frontiers in Cell and Developmental Biology, 8, 713.
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6

TNFα-Induced Inflammation Modulation

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For TNFα stimulation, cells were (unless otherwise indicated) treated with 10 ng/ml human recombinant TNFα (PeproTech EC Ltd., London, UK, in 0.1% BSA (cell culture grade, Sigma-Aldrich) in PBS (Sigma-Aldrich)) for 24 h. For immunocytochemical staining, cells were treated with TNFα for 15 min prior to fixation. All cells treated with TLR4 ligands (peptides and LPS) or NF-κB inhibitors were serum starved for 4 h before stimulation for 48 h with normal cultivation medium comprising respective ligands: 1 μg/ml (unless stated otherwise) ultrapure LPS derived from Salmonella minnesota (ultrapure S. minnesota R595 in endotoxin-free water, InvivoGen, Toulouse, France) or Escherichia coli (ultrapure E. coli LPS in endotoxin-free water, InvivoGen); 0.1–100 μg/ml fibrinogen from human plasma (50–70%, Sigma-Aldrich, dissolved in DMEM High Glucose and sterile filtered); 50–1000 ng/ml high mobility group box 1 (human recombinant, carrier-free HMGB1, BioLegend Ltd., London, UK, dissolved in 0.1% BSA in PBS); 0.001–10 μM amyloid-β peptide [1-42] (Aβ, rPeptide, Suffolk, UK, prepared in DMEM High Glucose) or heated Aβ (stock solution boiled at 90°C for 45 min); and 0.5–2.5 μM Bay11-7082 (Sigma-Aldrich, in DMSO (cell culture grade, Apollo Scientific)) or IMD0354 (10 mM, Sigma-Aldrich, in DMSO).
Zeuner M.T., Vallance T., Vaiyapuri S., Cottrell G.S, & Widera D. (2017). Development and Characterisation of a Novel NF-κB Reporter Cell Line for Investigation of Neuroinflammation. Mediators of Inflammation, 2017, 6209865.
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7

Optimizing Cell Migration Assays

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Migration assays were performed in a migration chamber (Millipore, Burlington, MA) according to the manufacturer’s directions using epidermal KCs, Swiss mouse 3T3 cells (ATCC, Manassas, VA), HMGB1 (BioLegend, San Diego, CA; #764004), or SDF1a (R&D Systems, Minneapolis, MN; Cat# 460-SD/CF) as bait. Positive controls were epidermal KCs, and negative controls were included medium without serum or growth factors. In addition, to confirm the specificity of HMGB1 as bait, HMGB1 antibody (Invitrogen, Carlsbad, CA; #PA5-29604) was added in the presence of HMGB1 (10 and 50 ng/ml), and non-antibody-treated HMGB1 group served as a positive control. Preliminary experiments were performed to establish optimum dosage and migration times, and experiments were conducted with triplicate wells and were repeated three times.
Park H., Lad S., Boland K., Johnson K., Readio N., Jin G., Asfaha S., Patterson K.S., Singh A., Yang X., Londono D., Singh A., Trempus C., Gordon D., Wang T.C, & Morris R.J. (2018). Bone marrow-derived epithelial cells and hair follicle stem cells contribute to development of chronic cutaneous neoplasms. Nature Communications, 9, 5293.
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8

Ligation of DsDna with HMGB1

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Cleaned 300 bp dsDNA with the 4 base 5′ overhanging ends was created as above and treated with T4 ligase (New England Biolabs, cat. # M0202), either in the absence or presence of HMGB1 (Biolegend, cat. # 557804), using the manufacturer’s recommended protocol (24 h at 37 °C). Ligase reactions were stopped with Proteinase K treatment (1 h at 37 °C, New England Biolabs, cat. # P8111) and then cleaned using the QIAquick PCR clean-up kit (Qiagen, cat. # 28104). Linear species remaining in the sample were removed using either T5 or T7 exonuclease (New England Biolabs, cat. # M0363, M0263) using the manufacturer’s recommended protocol.
Dent S.E., King D.P., Osterberg V.R., Adams E.K., Mackiewicz M.R., Weissman T.A, & Unni V.K. (2021). Phosphorylation of the aggregate-forming protein alpha-synuclein on serine-129 inhibits its DNA-bending properties. The Journal of Biological Chemistry, 298(2), 101552.
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9

HMGB1 Regulates vSMC Proliferation

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Cell proliferation was evaluated by Cell Counting Kit-8 (CCK-8) assay and BrdU incorporation assay. vSMCs transfected with shSox9 or siAtg5 were treated or without 100 mg/mL HMGB1 (BioLegend, USA) for indicated time. Afterward, the cells were incubated with CCK-8 solution (Dojindo, Kumamoto, Japan) or BrdU-labeling solution (Biovision, California, USA), and the absorbance at 450nm was detected using a microplate reader.
Yu Q., Liu J.X., Zheng X., Yan X., Zhao P., Yin C., Li W, & Song Z. (2022). Sox9 mediates autophagy-dependent vascular smooth muscle cell phenotypic modulation and transplant arteriosclerosis. iScience, 25(10), 105161.
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10

Western Blot Protein Expression Analysis

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Cell pellets were lysed in RIPA buffer (Emd Millipore, 20–188) with complete protease (Sigma, 11836153001) and phosphatase (Sigma, 04906837001) inhibitor cocktails. Lysates were collected by centrifugation, 18,000 × g for 15 min at 4 °C. Protein concentration was measured using Bradford BCA colorimetric assay (Bio-Rad, 5000006). In all, 15 µg of sample lysates were subjected to western blot analysis using 10% Tris-Glycine gel under reducing conditions. Proteins were transferred onto PVDF membranes (Emd Millipore, IPVH00010) and probed with the following primary antibodies: COX-2 [74 kDa] at 1:1000 (Cell Signaling Technologies, 12282 S); GAPDH [~37 kDa] at 1:2000 (Santa Cruz biotechnology, SC-32233); and HMGB1 [~29 kDa] at 1:1000 (Biolegend, 651402). Secondary antibodies were purchased from the following sources: anti-mouse-HRP at 1:10,000 (Boster, BA1075) and anti-rabbit-HRP at 1:10,000 (Cell Signaling Technologies, 7074 S). Western blot bands were visualized using an enhanced chemiluminescence system (Thermo, 32106) on the iBright™ CL750 system and iBright Analysis Software version 1.5.0.
Nikolos F., Hayashi K., Hoi X.P., Alonzo M.E., Mo Q., Kasabyan A., Furuya H., Trepel J., Di Vizio D., Guarnerio J., Theodorescu D., Rosser C., Apolo A., Galsky M, & Chan K.S. (2022). Cell death-induced immunogenicity enhances chemoimmunotherapeutic response by converting immune-excluded into T-cell inflamed bladder tumors. Nature Communications, 13, 1487.
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