Journal: International Journal of Molecular Medicine

Article Title: Bacterial lipopolysaccharide and antimicrobial LL-37 enhance ICAM-1 expression and NF-κB p65 phosphorylation in senescent endothelial cells

doi: 10.3892/ijmm.2019.4294

Figure Lengend Snippet: Role of FPR2 and P2X7 receptors in senescent and non-senescent endothelial cells. (A) Non-senescent HUVECs were preincubated with WRW4 (0.1 or 1 µ M) or KN-62 (0.1 or 1 µ M) for 30 min, and then incubated with LL-37 (5 µ g/ml) for 24 h. Alternatively, non-senescent HUVECs were preincubated with a combination of WRW4 (0.1 µ M) and KN-62 (0.1 µ M) for 30 min, and then incubated with LL-37 (5 µ g/ml) for 24 h. ICAM-1 protein expression levels were analyzed by western blotting. Relative expression of ICAM-1/GAPDH was calculated as a ratio to LL-37-stimulated cells without antagonists. Data are presented as the mean ± SD of at least three independent experiments. (B) Cell surface expression levels of LL-37 receptors FPR2 and (C) P2X7 were analyzed in senescent and non-senescent HUVECs by flow cytometry. Relative expression in senescent cells was calculated as a ratio to non-senescent cells. Data are presented as the mean ± SD of at least four independent experiments. * P<0.05, ** P<0.01 and *** P<0.001, with comparisons indicated by lines. FPR2, formyl peptide receptor 2; P2X7, purinergic receptor P2X 7; HUVECs, human umbilical vein endothelial cells; ICAM-1, intercellular adhesion molecule-1; Cont, control; NS, not significant.

Article Snippet: The FPR2 antagonist WRW4 peptide (sequence: WRWWWW) was from Alomone Labs; the P2X7 inhibitor KN-62 was from Sigma-Aldrich (Merck KGaA).

Techniques: Incubation, Expressing, Western Blot, Flow Cytometry, Control

Journal: International journal of oncology

Article Title: S100P regulates the collective invasion of pancreatic cancer cells into the lymphatic endothelial monolayer.

doi: 10.3892/ijo.2019.4812

Figure Lengend Snippet: Figure 6. S100P is involved in migration of LECs and CCID formation. (A) S100P mRNA expression in LECs following treatment with IL‑6 for 24 h. (B) LEC migration following treatment with IL‑6 (1 nM) was examined by Transwell migration assay. (C) S100P mRNA levels were significantly reduced following siRNA transfection (left panel). The IL‑6‑enhanced LEC migration was reduced following S100P knockdown (right panel). (D) CCID area fol lowing treatment with IL‑6 (1 nM). (E) LEC migration following treatment with culture supernatant from cancer cells, recombinant human S100P protein (1 nM) and RAGE antagonist peptide (1 µg/ml). (F) CCID area following treatment with recombinant human S100P protein (1nM) and RAGE antagonist peptide (1 µg/ml). (G) Comparison of S100P mRNA expression levels in cancer cells from single‑cell culture (normal plates) and from spheroid culture (low‑attachment plates) in two cell lines, BxPC3 and MIAPaCa2. *P<0.05 and ***P<0.001, with comparisons indicated by brackets. S100P, S100 calcium binding protein P; LECs, lymphatic endothelial cells; CCID, circular chemorepellent‑induced defect; IL, interleukin; si, small interfering; RAGE, receptor for advanced glycation end‑products; ctrl, control.

Article Snippet: To assess the effect of S100P on the migration of LECs, recombinant human S100P protein (Abcam), the receptor for advanced glycation end-products (RAGE) antagonist peptide (Tocris Bioscience, Bristol, UK), or recombinant human IL-6 protein (PeproTech, Inc., Rocky Hill, NJ, USA) was added to the lower chambers in 750 μl of EGM2MV medium.

Techniques: Migration, Expressing, Transwell Migration Assay, Transfection, Knockdown, Recombinant, Comparison, Binding Assay, Control

Journal: ImmunoHorizons

Article Title: Blood-Borne Microparticles Are an Inflammatory Stimulus in Type 2 Diabetes Mellitus

doi: 10.4049/immunohorizons.2200099

Figure Lengend Snippet: MPs production by murine neutrophils: cells were incubated with MPs from patients with DM+DFU or control subjects . Data show MPs produced by 1.8 × 10 5 murine neutrophils incubated with 10,000 blood-borne MPs from four subjects who each had DM and a DFU (two men, two women, age 57 ± 7.8 [SD] years, DM duration 24 ± 7.8 y, hemoglobin A1C 8.1 ± 0.8%) and four healthy, nondiabetic subjects (2 men, 2 women, age 59 ± 9.4 y). Inset shows effects of inhibitors: 25 µmol SSO, a CD36 inhibitor; 10 µmol RAGE inhibitor (RAGE antagonist peptide; Tocris, Inc.), 10 µmol TAK242 (resatorvid), a TLR4 inhibitor; 5 µmol PS. Data are mean ± SD; n = 4 replicate studies; incubation of cells with inhibitors did not alter viability (data not shown).

Article Snippet: BioAegis Therapeutics (North Brunswick, NJ) provided recombinant human gelsolin, and RAGE inhibitor (RAGE antagonist peptide) was from Tocris (catalog number [cat#] 6259).

Techniques: Incubation, Control, Produced

Journal: ImmunoHorizons

Article Title: Blood-Borne Microparticles Are an Inflammatory Stimulus in Type 2 Diabetes Mellitus

doi: 10.4049/immunohorizons.2200099

Figure Lengend Snippet: MPs production in 60 min in response to additions of 10,000 semirigid beads a displaying PS and PC to 1.8 × 10 5 neutrophils

Article Snippet: BioAegis Therapeutics (North Brunswick, NJ) provided recombinant human gelsolin, and RAGE inhibitor (RAGE antagonist peptide) was from Tocris (catalog number [cat#] 6259).

Techniques:

Journal: ImmunoHorizons

Article Title: Blood-Borne Microparticles Are an Inflammatory Stimulus in Type 2 Diabetes Mellitus

doi: 10.4049/immunohorizons.2200099

Figure Lengend Snippet: Western blots of human and murine neutrophils. Images are representative blots, and numbers beneath images are mean band densities and SD ( n = 4 for human cell, 6 for murine), where for each study band densities were normalized to actin loading and then to the control value on individual blots. Bold numbers reflect those significantly different from control, p < 0.05, ANOVA. Human cells were obtained from control subjects or those with DM+DFU (labeled DM, left columns). Murine cells were obtained from wild type or CD36 KO mice. For ex vivo incubations, 1.8 × 10 5 human ( A ) or murine ( B ) neutrophils were incubated for 1 h with 5.5 or 20 mM glucose or, where indicated, 5.5 mM glucose with 10,000 Ph+MPs or Ph-negative MPs from patients with DM+DFU without or with recombinant human gelsolin (Ph+MPs+gel), or with 10,000 PS- or PC-expressing semirigid beads. Murine neutrophils were incubated for 20 h before study with siRNA, either a control that depletes no protein or sequences to specifically deplete RAGE, TLR4, or NOS1AP.

Article Snippet: BioAegis Therapeutics (North Brunswick, NJ) provided recombinant human gelsolin, and RAGE inhibitor (RAGE antagonist peptide) was from Tocris (catalog number [cat#] 6259).

Techniques: Western Blot, Control, Labeling, Ex Vivo, Incubation, Recombinant, Expressing

Journal: Angewandte Chemie (International ed. in English)

Article Title: Small Molecule Control of Intracellular Protein Levels Through Modulation of the Ubiquitin Proteasome System

doi: 10.1002/anie.201307761

Figure Lengend Snippet: Summary of IAP inhibitors including AT-406 (developed by Ascenta Therapeutics and the University of Michigan),[62] which is administered orally in Phase 1 trials for solid tumors and lymphoma, Genentech/Roche’s GDC-0152 which is administered intravenously and is in Phase I trials for metastatic malignancies,[6b, 63] and the bivalent TL32711 (administered intravenously) developed by Tetralogics Pharma.[6b, 58] LCL161 (Novartis), AEG35156 and AEG40826 (Aegera), and YM155 (Astellas Pharma) are also in clinical trials but are not shown.[6b, 58] Selected SMAC mimics such as SM-122 and MV1 are also shown but are not in clinical trials.

Article Snippet: [ 60 ] An additional peptidic IAP antagonist is MV1, developed by Genentech.

Techniques: Clinical Proteomics

Journal: International Journal of Molecular Sciences

Article Title: Extracellular Vesicles as Biomarkers in Liver Disease

doi: 10.3390/ijms232416217

Figure Lengend Snippet: Clinical studies on EVs as biomarkers in patients with liver disease.

Article Snippet: Hepatobiliary Tumors (HCC and CCA) , EpCAM, CD147, ASGPR, CD133, and annexin V , Liver cancer (n = 172); cirrhosis (n = 54); and control (n = 202) , Annexin V+ EpCAM+ CD147+ taMPs were elevated in liver cancer (HCC and CCA). Annexin V+ EpCAM+ ASGPR1+ taMPs were increased in liver cancer compared to patients with cirrhosis. Annexin V+ EpCAM+ ASGPR1+ CD133+ taMPs allowed the distinction of liver malignancies. , Flow cytometry , Julich-Haertel H, et al. J Hepatol. 2017. [ ] .

Techniques: Marker, Control, Flow Cytometry, Clinical Proteomics, Centrifugation, Size-exclusion Chromatography, Isolation, In Vitro, Western Blot, Derivative Assay, Enzyme-linked Immunosorbent Assay, Biomarker Discovery, Acid Assay, Purification, Fluorescence, Microscopy, Transmission Assay, Electron Microscopy, Infection, Virus, Concentration Assay, Selection, Diagnostic Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Extracellular HMGB1 blockade inhibits tumor growth through profoundly remodeling immune microenvironment and enhances checkpoint inhibitor-based immunotherapy

doi: 10.1136/jitc-2020-001966

Figure Lengend Snippet: Glycyrrhizin, RAP, A box and EP exert efficient neutralizing effects on extracellular HMGB1 without altering tumor cell proliferation and apoptosis/necrosis. (A) Schematic representation of different modes of inhibition for extracellular HMGB1. Mouse RAW 264.7 cells were stimulated with recombinant HMGB1 (B) or conditioned media from 4T1 basal-like breast cancer cells (C) in the absence or presence of glycyrrhizin, RAP or a box (several concentrations were tested). Note the significant decrease of HMGB1-induced TNFα secretion when HMGB1 inhibitors were added in the cell cultures, indicating their efficient neutralizing effect. (D) EP was directly added in the culture medium of 4 different mouse basal-like breast cancer cell lines (4T1, 67NR, EpRas and EpH4). Forty-eight hours later, HMGB1 concentrations were determined by ELISA and the ability of EP to inhibit HMGB1 release in a dose-dependent manner was highlighted. (E) Oxygen consumption rate (OCR) and (F) extracellular acidification rate (ECAR) in 4T1 cells in the absence or presence of glycyrrhizin (1 nM), RAP (10 µM) and a box (0.5 µg/mL) were determined using Seahorse extracellular flux analyzer. No modification of OCR/ECAR was detected with these three HMGB1 inhibitors. (G) OCR and (H) ECAR in 4T1 cells following EP addition (concentration range: 0.1–10 mM). Histograms representing OCR (I) and ECAR (J) before (baseline) and after (stressed) oligomycin and FCCP addition in the absence or presence of EP. Both OCR and ECAR were strongly decreased with 5 and 10 mM EP. No significant change was detected with lower concentrations (0.1–1 mM). (K) Cell proliferation and (L) apoptosis of mouse 4T1 cells cultured without or with HMGB1 inhibitors (glycyrrhizin (1 nM), RAP (10 µM), a box (0.5 µg/mL) and EP (1 mM)) were determined using IncuCyte live cell analyzing system and annexin V-propidium iodide staining assay, respectively. No significant change was reported. The means±SEM (plus each individual data point) for at least three independent experiments are represented. Asterisks indicate statistically significant differences (*p<0.05; **p<0.01; ***p<0.001). P values were determined using one-way ANOVA, followed by Dunnett’s multiple comparison post-test (B, C, D, I, J, K, L). ANOVA, analysis of variance; ECAR, extracellular acidification rate; EP, ethyl pyruvate; HMGB1, high-mobility group box 1; RAGE, receptor for advanced glycation endproducts; RAP, RAGE antagonist peptide; TNFα, tumor necrosis factor-α.

Article Snippet: The neutralizing activity of glycyrrhizin (direct HMGB1 inhibitor) (Sigma Aldrich) as well as RAGE antagonist peptide (RAP) (Merck Millipore, Burlington, Massachusetts, USA) and truncated N-terminal domain of HMGB1 (A box) (HMGBiotech, Milan, Italy) was indirectly determined through analyzing their ability to attenuate HMGB1-induced TNFα release by macrophages/monocytes.

Techniques: Inhibition, Recombinant, Enzyme-linked Immunosorbent Assay, Modification, Concentration Assay, Cell Culture, Staining

Journal: Journal for Immunotherapy of Cancer

Article Title: Extracellular HMGB1 blockade inhibits tumor growth through profoundly remodeling immune microenvironment and enhances checkpoint inhibitor-based immunotherapy

doi: 10.1136/jitc-2020-001966

Figure Lengend Snippet: Extracellular HMGB1 blockade inhibits the growth of pre-established solid tumors in immunocompetent mice through activating anticancer immune responses. (A) Mouse basal-like breast cancer cells (4T1, 67NR and EpRas) were orthotopically injected into the mammary fat pad of immunocompetent BALB/c mice. Tumor-bearing mice were then treated at 3-day intervals with PBS (control) or HMGB1 inhibitors (glycyrrhizin (1 nM/kg), RAP (10 µM/kg), a box (500 µg/kg) and EP (1 mM/kg)). The mean tumor volumes±SEM are represented. (B) HMGB1 inhibitors were tested in nude mice implanted with 67NR cells. Note the absence of beneficial effect in these latter immunocompromised mice, indicating the dependence on the adaptive immune responses. (C) At day 17, 19 or 20 (depending on the analyzed cell line), tumors were harvested, CD45 + immune cells were isolated and analyzed by flow cytometry. The proportions of each analyzed immune cell population in both control and treated groups (pooled results) are shown. Note the drastic reduction of MDSC following extracellular HMGB1 blockade. (D) Total number of (CD45 + ) immune cells per milligram of tumor in both control and treated groups. (E) Scatter dot plots showing the percentage of each individual immune cell population (DC, PDC, CD4 + and CD8 + T cells, monocytic and granulocytic MDSC, neutrophils, M1 and M2 macrophages) among CD45 + cells in the different treatment groups. an increased M1/M2 ratio of macrophages was observed in most HMGB1 inhibitor-treated tumors. The intratumoral immune cells were analyzed in five mice per condition. (F) Scatter dot plots illustrating the percentage of tumor-infiltrating Treg (Foxp3 + ) CD4 + and CD8 + cells among total CD4 + and CD8 + populations in the different treatment groups. (G) Scatter dot plots illustrating the percentage of tumor-infiltrating PD-1 + CD4 + and PD-1 + CD8 + cells among total CD4 + and CD8 + populations in the treatment groups. The activation status of both DC (H) and PDC (I) in the different treatment groups was also determined by analyzing the expression of several cell surface markers (CD80, CD86, I-A/I-E, ILT3 and ICOSL). Data represent the mean fluorescent intensity (MFI)±SEM of 5 independent experiments in each group (each individual data point is shown). The number of apoptotic cancer cells (cleaved caspase 3 + ) (J) as well as the density of blood vessels within tumor microenvironment (CD31 + ) (K) were determined by computerized counting (using QuPath software). The number of cleaved caspase 3 + cells and the percentage of CD31 + pixels were reported to tumor area. The scale bar represents 100 µm. Asterisks indicate statistically significant differences (*p<0.05; **p<0.01; ***p<0.001). P values were determined using one-way ANOVA followed by Dunnett’s multiple comparison post-test (A, B, D, E, F, G, H, I) and (Welch-corrected) unpaired t-test (J, K). ANOVA, analysis of variance; DC, dendritic cell; EP, ethyl pyruvate; HMGB1, high-mobility group box 1; i.p, intraperitoneal; MDSC, myeloid-derived suppressor cells; pDC, plasmacytoid DC; RAP, RAGE antagonist peptide.

Article Snippet: The neutralizing activity of glycyrrhizin (direct HMGB1 inhibitor) (Sigma Aldrich) as well as RAGE antagonist peptide (RAP) (Merck Millipore, Burlington, Massachusetts, USA) and truncated N-terminal domain of HMGB1 (A box) (HMGBiotech, Milan, Italy) was indirectly determined through analyzing their ability to attenuate HMGB1-induced TNFα release by macrophages/monocytes.

Techniques: Injection, Isolation, Flow Cytometry, Activation Assay, Expressing, Software, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Extracellular HMGB1 blockade inhibits tumor growth through profoundly remodeling immune microenvironment and enhances checkpoint inhibitor-based immunotherapy

doi: 10.1136/jitc-2020-001966

Figure Lengend Snippet: Combination of anti-PD-L1 with HMGB1 inhibitors strongly inhibits tumor growth in syngeneic mouse models of basal-like breast cancer. (A) mRNA level of PD-L1 ( CD274 gene) in the four major molecular subtypes of breast cancer was determined using the METABRIC public dataset. (B) Representative example of breast cancer stained for PD-L1. Positive signals were detected on cancer cells and/or on inflammatory cells within tumor microenvironment. Semiquantitative evaluation of PD-L1 immunoreactivity (negative or >1% membrane staining) displayed by cancer cells (C) or inflammatory cells infiltrating the tumor microenvironment (D). The analyzed cancer specimens were categorized into the four molecular subtypes of breast cancer (LumA, LumB, HER2 + and basal-like). (E) The percentage of PD-L1 + cells in both epithelial/cancer (CD45 - ) and inflammatory (CD45 + ) components of untreated harvested 4T1/67NR tumors was determined by flow cytometry. Note the distinct profile displayed by these two cell lines. (F) Mouse breast cancer cells (4T1 and 67NR) were orthotopically injected into the mammary fat pad of immunocompetent BALB/c mice. Anti-PD-L1 antibody was tested alone (i.p. injection of 100 µg at days 4, 7 and 11) and in combination with HMGB1 inhibitors (RAP (10 µM/kg) and EP (1 mM/kg), treatment at 3-day intervals). The mean tumor volumes±SEM are represented. (G) The total number of (CD45 + ) immune cells per milligram of tumor was determined in the different treatment groups by flow cytometry. (H) Scatter dot plots illustrating the percentage of each individual immune cell population (DC, PDC, CD4 + and CD8 + T cells, monocytic and granulocytic MDSC, neutrophils, M1 and M2 macrophages) among CD45 + cells in both control and treated groups. The intratumoral immune cell infiltration was analyzed in five mice per condition. (I) scatter dot plots showing the percentage of tumor-infiltrating Treg (Foxp3 + ) CD4 + and CD8 + cells among total CD4 + and CD8 + populations in the different treatment groups. The activation status of DC (J) and pDC (K) was determined by flow cytometry. the expression of several surface markers (CD80, CD86, I-A/I-E, ILT3 and ICOSL) was assessed. Data represent the mean fluorescent intensity (MFI)±SEM of five independent experiments in each group (each individual data point is shown). (L) The apoptotic cancer cells (cleaved caspase 3 + ) were detected by immunohistochemistry and quantified using QuPath software. The number of positive cells was reported to tumor area (mm 2 ). The scale bar represents 100 µm. Asterisks indicate statistically significant differences (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). P values were determined using one-way ANOVA, followed by Bonferroni post-test (A, L), Fisher’s exact test (C, D) and one-way ANOVA followed by Dunnett’s multiple comparison post-test (F, G, H, I, J, K). ANOVA, analysis of variance; DC, dendritic cell; EP, ethyl pyruvate; HMGB1, high-mobility group box 1; i.p, intraperitoneal; METABRIC, Molecular Taxonomy of Breast Cancer International Consortium; pDC, plasmacytoid DC; RAP, RAGE antagonist peptide.

Article Snippet: The neutralizing activity of glycyrrhizin (direct HMGB1 inhibitor) (Sigma Aldrich) as well as RAGE antagonist peptide (RAP) (Merck Millipore, Burlington, Massachusetts, USA) and truncated N-terminal domain of HMGB1 (A box) (HMGBiotech, Milan, Italy) was indirectly determined through analyzing their ability to attenuate HMGB1-induced TNFα release by macrophages/monocytes.

Techniques: Staining, Flow Cytometry, Injection, Activation Assay, Expressing, Immunohistochemistry, Software

Journal: Journal for Immunotherapy of Cancer

Article Title: Extracellular HMGB1 blockade inhibits tumor growth through profoundly remodeling immune microenvironment and enhances checkpoint inhibitor-based immunotherapy

doi: 10.1136/jitc-2020-001966

Figure Lengend Snippet: Extracellular HMGB1 blockade enhances anti-PD-1-induced inhibition of tumor growth in vivo. (A) PD-1 mRNA expression ( PDCD1 gene) in the four major molecular subtypes of breast cancer was determined using the METABRIC public dataset. (B) Representative example of breast cancer stained for PD-1. Positive cells were observed in the epithelial component of the tumor as well as in the stroma surrounding cancer cells. (C) PD-1 + cell infiltration within tumor microenvironment was determined by computerized counting. Each point represents the number of positive cells/mm 2 for one independent tumor specimen. (D) Mouse breast cancer cells (4T1 and 67NR) were orthotopically injected into the mammary fat pad of immunocompetent BALB/c mice. Anti-PD-1 antibody was tested alone (i.p. injection of 200 µg at days 4, 7 and 11) and in combination with HMGB1 inhibitors (RAP (10 µM/kg) and EP (1 mM/kg), treatment at 3 day intervals). In parallel, the anticancer efficacy of these combination regimens was also compared with that displayed by each individual HMGB1 inhibitor used in monotherapy. The mean tumor volumes±SEM are represented. (E) The apoptotic cancer cells (cleaved caspase 3 + ) were detected by immunohistochemistry and quantified using QuPath software. The number of positive cells was reported to tumor area (mm 2 ). (F) The total number of (CD45 + ) immune cells per milligram of tumor was determined in the different treatment groups. (G) Scatter dot plots illustrating the percentage of each individual immune cell population (DC, PDC, CD4 + and CD8 + T cells, monocytic and granulocytic MDSC, neutrophils, M1 and M2 macrophages) among CD45 + cells in both control and treated groups. Reduced densities of granulocytic MDSC as well as an increase of M1 macrophages were especially observed in case of combination therapy. The intratumoral immune cells were analyzed in five mice per condition. (H) Scatter dot plots showing the percentage of tumor-infiltrating Treg (Foxp3 + ) CD4 + and CD8 + cells among total CD4 + and CD8 + populations in the different treatment groups. the activation status of DC (I) and pDC (J) was determined by flow cytometry. the expression of several surface markers (CD80, CD86, I-A/I-E, ILT3 and ICOSL) was analyzed. Data represent the mean fluorescent intensity (MFI)±SEM of 5 independent experiments in each group (each individual data point is shown). The scale bar represents 100 µm. Asterisks indicate statistically significant differences (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). P values were determined using one-way ANOVA followed by Bonferroni post-test (A, C, E) or Dunnett’s multiple comparison post-test (D, F, G, H, I, J). ANOVA, analysis of variance; DC, dendritic cell; HMGB1, high-mobility group box 1; METABRIC, Molecular Taxonomy of Breast Cancer International Consortium; MDSC, myeloid-derived suppressor cells; pDC, plasmacytoid DC; RAP, RAGE antagonist peptide.

Article Snippet: The neutralizing activity of glycyrrhizin (direct HMGB1 inhibitor) (Sigma Aldrich) as well as RAGE antagonist peptide (RAP) (Merck Millipore, Burlington, Massachusetts, USA) and truncated N-terminal domain of HMGB1 (A box) (HMGBiotech, Milan, Italy) was indirectly determined through analyzing their ability to attenuate HMGB1-induced TNFα release by macrophages/monocytes.

Techniques: Inhibition, In Vivo, Expressing, Staining, Injection, Immunohistochemistry, Software, Activation Assay, Flow Cytometry, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Extracellular HMGB1 blockade inhibits tumor growth through profoundly remodeling immune microenvironment and enhances checkpoint inhibitor-based immunotherapy

doi: 10.1136/jitc-2020-001966

Figure Lengend Snippet: A significant fraction of HMGB1 contained in tumor-extruded fluids is in its oxidized form and displays RAGE-dependent tolerogenic properties. (A, B) The ROS accumulation in both breast (4T1, 67NR, EpRas) and lung (TC-1) cancer cells used in the present study was assessed by flow cytometry. N-acetylcysteine (5 mM) and tert-butyl hydroperoxide (100 µM) were used as negative and positive controls, respectively. Results represent the means±SEM of four independent experiments (each individual data point is shown). (C) The redox state of extracellular HMGB1 contained in tumor-extruded fluids was analyzed by Western blot. All samples were directly alkylated in order to ‘freeze’ the redox state of HMGB1 molecules. Recombinant HMGB1 (0.5 µg) incubated with either H 2 O 2 or DTT (and then alkylated) were used as controls. (D) Oxidized/reduced-disulfide HMGB1 ratio (%) was calculated from the Western blot bands using ImageJ software. (E) DCs were incubated with terminally oxidized, fully reduced or disulfide HMGB1 for 24 hours before being stimulated with LPS for 24 hours. The expression of cell-surface molecules (CD80, CD83, CD86, HLA-DR, HLA-ABC and CCR7) was then measured by flow cytometry. All data were normalized to LPS-stimulated DC. Data represent the relative mean fluorescent intensity (MFI)±SEM of at least five independent experiments (each individual data point is shown). (F) DCs were incubated with terminally oxidized HMGB1 for 24 hours before being stimulated with LPS for 24 hours. When indicated, an inhibitor of RAGE (10 µM RAP) or TLR4 (2 µM LPS-RS) was added in the cell culture. The expression of DC activation markers was determined by flow cytometry. All data were normalized to LPS-stimulated DC. The relative MFI±SEM of 7 independent experiments are shown. Asterisks indicate statistically significant differences (*p<0.05, **p<0.01, ***p<0.001). P values were determined using one-way ANOVA, followed by Dunnett’s multiple comparison post-test (A, B, E, F). ANOVA, analysis of variance; DC, dendritic cell; HMGB1, high-mobility group box 1.

Article Snippet: The neutralizing activity of glycyrrhizin (direct HMGB1 inhibitor) (Sigma Aldrich) as well as RAGE antagonist peptide (RAP) (Merck Millipore, Burlington, Massachusetts, USA) and truncated N-terminal domain of HMGB1 (A box) (HMGBiotech, Milan, Italy) was indirectly determined through analyzing their ability to attenuate HMGB1-induced TNFα release by macrophages/monocytes.

Techniques: Flow Cytometry, Western Blot, Recombinant, Incubation, Software, Expressing, Cell Culture, Activation Assay