Journal: Open Life Sciences

Article Title: Aggravated renal fibrosis is positively associated with the activation of HMGB1-TLR2/4 signaling in STZ-induced diabetic mice

doi: 10.1515/biol-2022-0506

Figure Lengend Snippet: Activation of the HMGB1/TLR2/4 signaling pathway in the kidneys of STZ-induced diabetic mice. Mice were treated with STZ at 100 or 150 mg/kg by intraperitoneal injection. The kidney was dissected on Day 64. Immunohistochemical staining of HMGB1, TLR2, and TLR4 in the kidneys (a), photographed at 400× magnification. Protein expression of HMGB1 and p-NF-κB in the kidneys by Western blot (b), n = 3. PCNA and β-actin were used as internal controls. Data are expressed as the mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, vs the normal group, tested by one-way ANOVA and Fisher’s PLSD.

Article Snippet: After quenching endogenous peroxidase activity with 3% H 2 O 2 and blocking with 5% bovine albumin, the sections were incubated with antibodies recognizing HMGB1 (1:400, Proteintech), TLR2 (1:500, Arigo), TLR4 (1:400, Abcam), F4/80 (1:300, Servicebio), CD14 (1:500, Servicebio), Col IV (1:250, Abcam), and FN (1:250, Abcam) overnight.

Techniques: Activation Assay, Injection, Immunohistochemical staining, Staining, Expressing, Western Blot

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:

Journal: European Journal of Histochemistry : EJH

Article Title: Inhibition of HMGB1 suppresses inflammation and catabolism in temporomandibular joint osteoarthritis via NF-κB signaling pathway

doi: 10.4081/ejh.2022.3357

Figure Lengend Snippet: Inhibition of HMGB1 decreases the expression of pro-inflammatory cytokines and catabolic mediators in rat synovium of CFA induced TMJOA. A) The schematic chart for the timeline of treating rats. B) The expression of ADAMTS5, MMP13, IL-1β, IL-6 and negative controls was detected by immunohistochemistry in the synovium of CFA groups, CFA with anti-HMGB1 antibody treatment groups at day 21, or in the control synovium; scale bar: 100 μm. C) Quantitative analysis of ADAMTS5, MMP13, IL-1β and IL- 6 in the synovium of rats; data are shown as the means with 95% CI and analyzed by one-way analysis of variance (ANOVA), n=5, *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: After 2 weeks, 5 rats previously injected with CFA were intraperitoneally injected with 100 μg/kg/day anti HMGB1 antibody (MAB1690-100; R&D System) for 7 days.

Techniques: Inhibition, Expressing, Immunohistochemistry, Control

Journal: European Journal of Histochemistry : EJH

Article Title: Inhibition of HMGB1 suppresses inflammation and catabolism in temporomandibular joint osteoarthritis via NF-κB signaling pathway

doi: 10.4081/ejh.2022.3357

Figure Lengend Snippet: Inhibition of HMGB1 alleviates the damage of cartilage and subchondral bone in CFA induced TMJOA. A) H&E, Safranin O and Masson trichrome staining of rat models at day 21; scale bar: 100 μm. B-D) Analysis of the condylar cartilage thickness, the Safranin O positive areas and unmineralized bone areas; data are shown as the means with 95% CI and analyzed by one-way analysis of variance (ANOVA), n=5, *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: After 2 weeks, 5 rats previously injected with CFA were intraperitoneally injected with 100 μg/kg/day anti HMGB1 antibody (MAB1690-100; R&D System) for 7 days.

Techniques: Inhibition, Staining

Journal: European Journal of Histochemistry : EJH

Article Title: Inhibition of HMGB1 suppresses inflammation and catabolism in temporomandibular joint osteoarthritis via NF-κB signaling pathway

doi: 10.4081/ejh.2022.3357

Figure Lengend Snippet: Inhibition of HMGB1 decreases the expression of pro-inflammatory cytokines and catabolic mediators in rat cartilage of CFA induced TMJOA. A) The expression of ADAMTS5, MMP13, IL-1β, IL-6 and negative controls was detected by immunohistochemistry in the cartilage of CFA groups, CFA with anti-HMGB1 antibody treatment groups at day 21, or in the control cartilage; scale bar: 100 μm. B) Rate of ADAMTS5, MMP13, IL-1β and IL-6 positive cells in the cartilage was markedly increased in the CFA groups, while markedly inhibited after treatment with anti-HMGB1 antibody; data are shown as the means with 95% CI and analyzed by one-way analysis of variance (ANOVA), n=5, *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: After 2 weeks, 5 rats previously injected with CFA were intraperitoneally injected with 100 μg/kg/day anti HMGB1 antibody (MAB1690-100; R&D System) for 7 days.

Techniques: Inhibition, Expressing, Immunohistochemistry, Control

Journal: European Journal of Histochemistry : EJH

Article Title: Inhibition of HMGB1 suppresses inflammation and catabolism in temporomandibular joint osteoarthritis via NF-κB signaling pathway

doi: 10.4081/ejh.2022.3357

Figure Lengend Snippet: Inhibition of HMGB1 suppresses the NF-κB signaling pathway in vivo. Expression of phospho-p65 in the synovium (A) and cartilage (B) of CFA groups, CFA with anti-HMGB1 antibody treatment groups at day 21 were detected by IF staining, or in the control groups; data are shown as the means with 95% CI and analyzed by one-way analysis of variance (ANOVA), n=5, *p<0.05, **p<0.01, ***p<0.001; scale bar: 100 μm. C) Schematic diagram of inhibition of HMGB1 suppresses inflammation and catabolism in temporomandibular joint osteoarthritis via NF-κB signaling pathway.

Article Snippet: After 2 weeks, 5 rats previously injected with CFA were intraperitoneally injected with 100 μg/kg/day anti HMGB1 antibody (MAB1690-100; R&D System) for 7 days.

Techniques: Inhibition, In Vivo, Expressing, Staining, Control

Journal: Journal of Inflammation Research

Article Title: Adiponectin Ameliorates Hyperglycemia-Induced Retinal Endothelial Dysfunction, Highlighting Pathways, Regulators, and Networks

doi: 10.2147/jir.s358594

Figure Lengend Snippet: Figure 4 (A–C) Canonical pathways by IPA core analysis. (A) Histogram displays the most relevant canonical pathways (p <0.05) involved in the response of HRMECs cells to APN treatment. The rank was based on the log p-value, the ratio of genes of the dataset compared to the knowledge base of IPA. See supplementary tables for details. (B) Bars display the values of Z- activation of the most relevant canonical pathways. (C) Network displays the HMGB1 pathway cascade and involves the downstream genes that are differentially expressed; downregulated as the green color and upregulated as red color, labeled with values of fold changes, and their role in biological process. Abbreviations: A, activation; p, phosphorylation; e, expression; c, causative to; and PP, protein-binding. See legends for details.

Article Snippet: In addition, an aliquot of each supernatant was assayed in duplicate as per the manufacturer’s instruction for IL-8, TNF-α, and IL-Iβ using the human cytokine multiplex immunoassay kit (HADK2MAG-61) from Millipore (Merck Millipore, Billerica, MA, USA) as published previously.16 Western Blot Proteins were extracted using RIPA buffer, and the protein concenteration was evaluated using BCA assay then resolved on 10% Bis-Tris gels (NuPAGE, Novex, Thermo Fischer), transferred to PVDF membrane, and immunoblotted using Primary antibodies HMGB1 (sc-548457), AdipoR1 (sc-518030), and AdipoR2 (sc-514045)(purchased from Santa Cruz, Germany), and SOD2 (cst#13141), and Beta-Actin (cst#3700) (purchased from cell signaling, USA).

Techniques: Activation Assay, Labeling, Phospho-proteomics, Expressing, Protein Binding

Journal: Frontiers in Cellular Neuroscience

Article Title: Receptor for Advanced Glycation End Products and its Inflammatory Ligands are Upregulated in Amyotrophic Lateral Sclerosis

doi: 10.3389/fncel.2015.00485

Figure Lengend Snippet: Expression of RAGE and its ligands in control and ALS thoracic spinal cord tissue. (A) RAGE expression in control ( A , top), and in ALS tissue ( A , bottom). (B) S100B immunostaining in control tissue ( B , top) and in ALS tissue ( B , bottom). (C) HMGB1 immunostaining in the control tissue ( C , top) and in ALS tissue ( C , bottom). (D) CML immunostaining in control spinal cord ( D , top) and in ALS spinal cord ( D , bottom). (E–G) Quantification of immunostaining intensity revealed that expression of all studied proteins was significantly increased in ALS thoracic spinal cord tissue compared to controls. S100B (E) was increased about 70%, HMGB1 (F) displayed almost threefold increase and CML (G) showed almost double level of increase in immunostaining between ALS and control subjects. Sections are representative of n = 6 control and n = 5 ALS tissue samples per condition. Error bars represent mean ± SEM, ∗ p < 0.05. Scale bar: 50 μm.

Article Snippet: Tissue homogenates (30 μg) were subjected to electrophoresis using 4–12% SDS-PAGE gels and specific protein signals were detected using the following antibodies: mouse anti-β-actin (A1978, 1:4000, Sigma); rabbit anti-RAGE (GTX23611, 1:1000, GeneTex); rabbit anti-S100B (ab52642, 1:5000, Abcam); and rabbit anti-HMGB1 (GTX101277, 1:1000, GeneTex).

Techniques: Expressing, Immunostaining

Journal: Frontiers in Cellular Neuroscience

Article Title: Receptor for Advanced Glycation End Products and its Inflammatory Ligands are Upregulated in Amyotrophic Lateral Sclerosis

doi: 10.3389/fncel.2015.00485

Figure Lengend Snippet: High magnification images of immunostaining for RAGE and its ligands in the thoracic spinal cord. Increased immunostaining pattern on the border of gray (lamina IX) and white matter was observed for (A) RAGE, (B) S100B, (C) HMGB1 and (D) CML in ALS versus control samples. Scale bar: 50 μm.

Article Snippet: Tissue homogenates (30 μg) were subjected to electrophoresis using 4–12% SDS-PAGE gels and specific protein signals were detected using the following antibodies: mouse anti-β-actin (A1978, 1:4000, Sigma); rabbit anti-RAGE (GTX23611, 1:1000, GeneTex); rabbit anti-S100B (ab52642, 1:5000, Abcam); and rabbit anti-HMGB1 (GTX101277, 1:1000, GeneTex).

Techniques: Immunostaining

Journal: Frontiers in Cellular Neuroscience

Article Title: Receptor for Advanced Glycation End Products and its Inflammatory Ligands are Upregulated in Amyotrophic Lateral Sclerosis

doi: 10.3389/fncel.2015.00485

Figure Lengend Snippet: Co-expression of RAGE and RAGE ligands S100B, CML, and HMGB1 is higher in human ALS spinal cord. (A) Triple staining for RAGE (red), S100B (green), CML (blue) revealed increased immunoexpression of these proteins in the ALS spinal cord ( A , right) as compared to controls ( A , left) and a high degree of RAGE/ligand overlapping was observed in ALS samples (merged images). (B) Expression of RAGE (red) and its ligands, S100B (green) and HMGB1 (blue) was highly increased in the ALS ( B , right) spinal cord as compared to controls ( B , left) and a high degree of RAGE/ligand co-expression observed in ALS samples (merged images); control ( n = 6) vs. ALS samples ( n = 5). Scale bar: 100 μm. (C) A schematic diagram showing different regions of spinal cord; for the purpose of the study we examined thoracic motor spinal cord ventral horn lamina IX and surrounding white matter.

Article Snippet: Tissue homogenates (30 μg) were subjected to electrophoresis using 4–12% SDS-PAGE gels and specific protein signals were detected using the following antibodies: mouse anti-β-actin (A1978, 1:4000, Sigma); rabbit anti-RAGE (GTX23611, 1:1000, GeneTex); rabbit anti-S100B (ab52642, 1:5000, Abcam); and rabbit anti-HMGB1 (GTX101277, 1:1000, GeneTex).

Techniques: Expressing, Staining

Journal: Frontiers in Cellular Neuroscience

Article Title: Receptor for Advanced Glycation End Products and its Inflammatory Ligands are Upregulated in Amyotrophic Lateral Sclerosis

doi: 10.3389/fncel.2015.00485

Figure Lengend Snippet: High magnification images of white/gray matter showing triple staining for RAGE and its ligands S100B, CML, and HMGB1. Immunostaining for RAGE (red) and its ligands S100B (green) and CML or HMGB1 (blue) revealed low immunoexpression in control tissue ( A and C ) and high immunoexpression in ALS tissue ( B and D ). Sections are representative of n = 6 control and n = 5 ALS tissue samples per condition. Scale bar: 100 μm.

Article Snippet: Tissue homogenates (30 μg) were subjected to electrophoresis using 4–12% SDS-PAGE gels and specific protein signals were detected using the following antibodies: mouse anti-β-actin (A1978, 1:4000, Sigma); rabbit anti-RAGE (GTX23611, 1:1000, GeneTex); rabbit anti-S100B (ab52642, 1:5000, Abcam); and rabbit anti-HMGB1 (GTX101277, 1:1000, GeneTex).

Techniques: Staining, Immunostaining

Journal: Frontiers in Cellular Neuroscience

Article Title: Receptor for Advanced Glycation End Products and its Inflammatory Ligands are Upregulated in Amyotrophic Lateral Sclerosis

doi: 10.3389/fncel.2015.00485

Figure Lengend Snippet: Protein levels of RAGE and its ligands S100B and HMGB1 are higher in human ALS spinal cord. Western blot analysis of RAGE (A) , RAGE ligands S100B (B) and HMGB1 (C) in control and ALS spinal cord tissue. The original blots were stripped followed by incubation with the other antigens under study. Signal for test antigen was then normalized to β-actin and the relative band densities were reported. n = 3 subjects/group. Error bars represent mean ± SEM, ∗ p < 0.05.

Article Snippet: Tissue homogenates (30 μg) were subjected to electrophoresis using 4–12% SDS-PAGE gels and specific protein signals were detected using the following antibodies: mouse anti-β-actin (A1978, 1:4000, Sigma); rabbit anti-RAGE (GTX23611, 1:1000, GeneTex); rabbit anti-S100B (ab52642, 1:5000, Abcam); and rabbit anti-HMGB1 (GTX101277, 1:1000, GeneTex).

Techniques: Western Blot, Incubation

Journal: Frontiers in Cellular Neuroscience

Article Title: Receptor for Advanced Glycation End Products and its Inflammatory Ligands are Upregulated in Amyotrophic Lateral Sclerosis

doi: 10.3389/fncel.2015.00485

Figure Lengend Snippet: A proposed mechanism of RAGE action in ALS spinal cord. We propose that during pathological processes in ALS, neuronal and microglial RAGE becomes activated by RAGE ligands such as AGEs, S100B, and HMGB1. Once activated, RAGE triggers a cascade of metabolic changes, contributing to the release of reactive oxygen species (ROS) and inflammatory cytokines, subsequently resulting in altered protein structures and misfolded protein accumulation, impaired mitochondrial function and growing energy deficits ultimately leading to neuronal dysfunction and apoptosis.

Article Snippet: Tissue homogenates (30 μg) were subjected to electrophoresis using 4–12% SDS-PAGE gels and specific protein signals were detected using the following antibodies: mouse anti-β-actin (A1978, 1:4000, Sigma); rabbit anti-RAGE (GTX23611, 1:1000, GeneTex); rabbit anti-S100B (ab52642, 1:5000, Abcam); and rabbit anti-HMGB1 (GTX101277, 1:1000, GeneTex).

Techniques:

Journal: Journal of Nanobiotechnology

Article Title: Combination of ferroptosis and pyroptosis dual induction by triptolide nano-MOFs for immunotherapy of Melanoma

doi: 10.1186/s12951-023-02146-0

Figure Lengend Snippet: A Relative cell viability of B16F10 cells after incubation in different preparation groups (TFBF, TPL, TPL@TFB and TPL@TFBF with and without various compounds). B Expression of Nrf2 and GPX4 proteins after different treatments. C Relative GSH levels in B16F10 cells after different preparations. Data are expressed as the mean ± SD (n = 3). ns, not significant, ****P < 0.0001. D Relative cell viability (compared to the group without inhibitor treatment) of B16F10 cells after different treatments in the presence of DEVD. E Expression of GSDME, GSDME-N and cleaved-caspase-3 proteins after different treatments. F LDH release from B16F10 cells after different treatments. Data are expressed as the mean ± SD (n = 3). ns, not significant, **P < 0.01, ****P < 0.0001. G Annexin V/PI double staining analysis of B16F10 cells treated with different preparations. H Annexin V + /PI + quantification of ( G ). Data are expressed as the mean ± SD (n = 3). **P < 0.01, ***P < 0.001, ****P < 0.0001. Release of IL-1β ( I ), ATP ( J ) and ( K ) HMGB1 from B16F10 cells after treatment with different preparations. L Expression of the HMGB1 protein in B16F10 cells after different treatments. Data are presented as the mean ± SD (n = 3). ***P < 0.001, ****P < 0.0001. M Flow cytometric results of CRT exposure from B16F10 cells treated with different agents and ( N ) quantitative average fluorescence intensity. Data are expressed as the mean ± SD (n = 3). *P < 0.05, ***P < 0.001

Article Snippet: The antibodies of HMGB1, GPX4 and calreticulin polyclonal antibody were from Proteintech Group Inc. (Wuhan, China).

Techniques: Incubation, Expressing, Double Staining, Fluorescence