Journal: bioRxiv

Article Title: An immunomechanical checkpoint PYK2 governs monocyte-to-macrophage differentiation in pancreatic cancer

doi: 10.1101/2024.11.19.624405

Figure Lengend Snippet: Western blot analysis of PYK2 and p-PYK2 in THP-1 cells treated with PMA and Yoda1 (300 nM) in the presence or absence of EGTA (4 mM) for 6 hours. b, Evaluation of cytosolic Ca 2+ levels in THP-1 cells transfected with si-NC or si-PIEZO1. Following transfection of THP-1 cells with si-NC, si-PIEZO1_1, or si-PIEZO1_2, cells were primed with PMA for 48 hours. Stimuli Yoda1 was added into the culture medium at 1 min as indicated by a black arrow. Fluorescence intensities of calcium indicator Fluo-4 AM in individual cells were tracked over time. The relative fluorescence intensities (ΔF/F0) of Fluo-4 AM-loaded THP-1 cells were calculated (left), while the average maximum peak intensity (Fmax/F0) and the intensity at the steady state (F8/F0) were calculated at 7 min post Yoda1 stimulation from si-NC (58 cells), si-PIEZO1_1 (75 cells) and si-PIEZO1_2 (58 cells) treated cells (middle and right). c, Western blot analysis of PYK2 and p-PYK2 in THP-1 cells following PIEZO1 silencing. THP-1 cells were transfected with si-NC, si-PIEZO1_1 or si-PIEZO1_2, followed by treatment of PMA for 48 hours. d-e, Western blot analysis of PYK2 and p-PYK2 in THP-1 cells treated with PMA and anti-integrin β1 antibody (5 µg/ml), or inhibitors including PYK2 inhibitor VS-4718 (1 µM), F-actin polymerization inhibitor Latrunculin B (5 µM) and Myosin II inhibitor Blebbistatin (10 µM) for 24 hours. IgG antibody or DMSO served as controls. f, Western blot analysis of PYK2 in the nuclear or cytoplasmic fractions of suspended THP-1 cells and those primed with PMA for 24h and 48h, respectively. g, Investigation of the colocalization of PYK2 with the nucleus in suspended THP-1 cells and those primed with PMA for 24h and 48h, respectively. Nucleus were labeled with DAPI. Representative immunofluorescence images of PYK2 (green) and DAPI (blue) in THP-1 cells are presented (left). Histogram of the normalized fluorescent intensity of PYK2 and DAPI was obtained from the cross-sectional lines through cell nucleus (right). Scale bars, 10 μm. h, Schematic of CUT&Tag for investigating the chromatin recruitment of PYK2. i, CUT&Tag sequencing analysis of PYK2 in suspended THP-1 cells and those primed with PMA for 24h and 48h, respectively. j, The bar plot showing GO enrichment analysis of downstream genes associated with promoter regions (<1 kb) identified from CUT&Tag data in mechanically trained THP-1 cells (48h vs 0h). The color gradient represents the -log10 adjusted P value, and the x-axis indicates the GeneRatio. k, CUT&Tag tracks of ACTR3 and RELA loci in suspended THP-1 cells and those primed with PMA for 24h and 48h. l, qPCR validation of ACTR3 and RELA loci in suspended THP-1 cells and those primed with PMA for 24h and 48h. m , Relative mRNA expression of ACTR3 and RELA in suspended THP-1 cells and those primed with PMA for 24h and 48h. n, Relative mRNA expression of ACTR3 and RELA in THP-1 cells following PTK2B silencing. o, Relative mRNA expression of ACTR3 and RELA in WT or Ptk2b KO mouse progenitors primed with M-CSF for 3 days. p, Flow cytometry analysis of mouse progenitors treated with M-CSF and Arp2/3 inhibitor CK-666 (50, 100 and 200 µM) for 3 days. q, Illustration outlining the PYK2-mediated mechanotransduction and differentiation process of monocytes within a stiff microenvironment. Monocytes respond to biochemical cues by interacting with their environment, initiating the differentiation process. Upon adhesion to the rigid matrix, activation of Piezo1 and integrin occurs, leading to the activation of PYK2 and subsequent reorganization of F-actin. Following this, PYK2 translocates to the nucleus, and selectively binds to gene regulatory regions associated with actin cytoskeleton organization and NF-κB signaling, such as ACTR3 and RELA , and others, thereby promoting monocyte differentiation. P values in ( o ) were determined by two-tailed unpaired Student’s t-test; P values in ( b ), ( l ), ( m ), ( n ) and ( p ) were determined by ordinary one-way ANOVA. Data shown in ( i ), ( k ) and ( l ) are representative of two experiments. Data shown in ( b ), (m) , ( n ), ( o ) and ( p ) are representative of three experiments.

Article Snippet: Each group received either an IgG2a isotype control (clone 2A3, BioXCell, US) or αPD-1 antibody (anti-mouse PD1, clone RMP1-14, BioXCell, US) treatment via intraperitoneal (i.p.) injection every 4 days at a dose of 200 μg, as previously described .

Techniques: Western Blot, Transfection, Fluorescence, Labeling, Immunofluorescence, Sequencing, Expressing, Flow Cytometry, Activation Assay, Two Tailed Test

Journal: Cellular Microbiology

Article Title: IFITM3 ‐containing exosome as a novel mediator for anti‐viral response in dengue virus infection

doi: 10.1111/cmi.12339

Figure Lengend Snippet: Identification of IFITM 3‐containing exosomes derived from 29 3T and HUVEC cells. A–D. Cell culture supernatants of HUVEC or HepG2 cells transfected with IFITM3 siRNA ‐1 ( HUVEC ‐ siIFITM3 or HepG2 ‐ siIFITM3 ), from HUVEC or HepG2 transfected with non‐targeting control siRNA ( HUVEC‐siNC or HepG2‐siNC ), from293 T cells transfected with pc DNA 3 (293 T ‐ V ector), or from 293 T cells transfected with the pc DNA ‐ IFITM 1, 2, 3‐ F lag construct (293 T ‐ IFITM 1, 2, 3) for 48 h, were collected and concentrated for SDS‐PAGE and immunoblotting, using an anti‐ IFITM 3 antibody, with cell lysates as positive control. E. Electron micrographs of crude exosomes negatively stained with uranyl acetate and examined at 80 kV are shown. F. Purified IFITM 3‐containing exosomes derived from each group of cells above described were analysed by immunoblotting with anti‐ IFITM 3, anti‐flotillin‐2, anti‐ CD 63, anti‐calnexin (endoplasmic reticulum, ER marker) and anti‐ GM 130 (Golgi marker) antibodies. IFITM 3 is identified in the exosomes derived from each group of cells as indicated.

Article Snippet: Following a blocking step using the blocking buffer (Tris‐buffered saline, TBS, containing 5% non‐fat milk) for 1 h at room temperature, the membranes were incubated overnight at 4°C with the following specific primary antibodies: monoclonal anti‐IFITM1, monoclonal anti‐IFITM2, polyclonal anti‐IFITM3 (Proteintech Group, Chicago, IL), monoclonal anti‐Flag (Sigma‐Aldrich, St Louis, MO), D1‐11 (anti‐DENV‐2 E mouse monoclonal antibody; Santa Cruz Biotechnology, Santa Cruz, CA), monoclonal anti‐Actin (Sigma‐Aldrich), polyclonal anti‐CD63 (H‐193; Santa Cruz Biotechnology), anti‐Flotillin 2 (C‐terminal; Sigma‐Aldrich), anti‐calnexin (Proteintech Group), and monoclonal anti‐GM130 (Cell Signaling, Danvers, MA) antibodies.

Techniques: Derivative Assay, Cell Culture, Transfection, Construct, SDS Page, Western Blot, Positive Control, Staining, Purification, Marker

Journal: bioRxiv

Article Title: Acidosis, Zinc and HMGB1 in Sepsis: A Common Connection Involving Sialoglycan Recognition

doi: 10.1101/2020.07.15.198010

Figure Lengend Snippet: CD11b expression was determined by flow cytometry after incubating whole blood with/without HMGB1 (1 µg/ml). A) Neutrophils are activated when incubated with HMGB1 in whole blood at pH 7.2 (Chromatograms: Red-Control, Blue-whole blood at pH 7.5, Orange-whole blood at pH 7.2, Green-whole blood at pH 7.5 with HMGB1, Cyan-whole blood at pH 7.2 with HMGB1). B) Activation is partially attenuated with an HMGB1-blocking antibody (50 µg/ml) (Chromatograms: Red-Isotype control, Blue-whole blood at pH 7.2, Green-whole blood at pH 7.2 with HMGB1, Orange-whole blood at pH 7.2 with HMGB1 and an HMGB1-block

Article Snippet: Purified mouse anti-HMGB1 antibody (BioLegend; catalogue number-651402, Lot# B219634) and Cy3-conjugated goat anti-mouse IgG (Jackson ImmunoResearch; catalogue number-115-165-008) were used.

Techniques: Expressing, Flow Cytometry, Incubation, Control, Activation Assay, Blocking Assay

Journal: bioRxiv

Article Title: Acidosis, Zinc and HMGB1 in Sepsis: A Common Connection Involving Sialoglycan Recognition

doi: 10.1101/2020.07.15.198010

Figure Lengend Snippet: A) Ability of HMGB1 to bind to different cell types of the blood (erythrocytes, monocytes and neutrophils) was determined by using different concentrations (100 ng/ml, 500 ng/ml and 5 µg/ml) of HMGB1 at physiological conditions. B) Different cell types of blood were used for binding with HMGB1 (100 ng/ml) at physiological and lower pH (pH 7.2, adjusted with lactic acid).

Article Snippet: Purified mouse anti-HMGB1 antibody (BioLegend; catalogue number-651402, Lot# B219634) and Cy3-conjugated goat anti-mouse IgG (Jackson ImmunoResearch; catalogue number-115-165-008) were used.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Acidosis, Zinc and HMGB1 in Sepsis: A Common Connection Involving Sialoglycan Recognition

doi: 10.1101/2020.07.15.198010

Figure Lengend Snippet: A) 1 ml of blood was drawn from a healthy individual and spun down. The plasma was replaced with HEPES buffer containing zinc (500 µM of Zn 2+ ) or plasma was added back. The CD11b expression as a marker of neutrophil activation was measured. B) The binding of HMGB1 to α1-acid glycoprotein was checked with a binding buffer using different pH ranging from 7.1 to 7.8.

Article Snippet: Purified mouse anti-HMGB1 antibody (BioLegend; catalogue number-651402, Lot# B219634) and Cy3-conjugated goat anti-mouse IgG (Jackson ImmunoResearch; catalogue number-115-165-008) were used.

Techniques: Clinical Proteomics, Expressing, Marker, Activation Assay, Binding Assay

Journal: bioRxiv

Article Title: Acidosis, Zinc and HMGB1 in Sepsis: A Common Connection Involving Sialoglycan Recognition

doi: 10.1101/2020.07.15.198010

Figure Lengend Snippet: A) The sialoglycan array was performed to test the binding of HMGB1 with multiple sialylated probes. The binding buffer used for the assay either contained zinc and pH 7.5, no zinc, pH 7.5, with zinc, pH 7.2 and no zinc at pH 7.2. The concentration of zinc used was 15 µM and 150 µM. (Representative image of a single experiment. Wilcoxon matched pairs signed rank test used. **** represents p-value < 0.0001) B) Additional microarray experiments with 500 µM further resolve the pH-dependent binding difference. (Representative image of the mean of two experiments. Unpaired t-test with Welch’s correction used to compare the two groups. **** represents p-value < 0.0001) C) The difference of HMGB1 to sialosides and non-sialosides at physiological pH in the presence of 500 µM zinc. (Representative image of a single experiment where zinc was used at a concentration of 500 µM. Kolmogorov-Smirnov test used. **** represents p-value < 0.0001)

Article Snippet: Purified mouse anti-HMGB1 antibody (BioLegend; catalogue number-651402, Lot# B219634) and Cy3-conjugated goat anti-mouse IgG (Jackson ImmunoResearch; catalogue number-115-165-008) were used.

Techniques: Binding Assay, Concentration Assay, Microarray

Journal: bioRxiv

Article Title: Acidosis, Zinc and HMGB1 in Sepsis: A Common Connection Involving Sialoglycan Recognition

doi: 10.1101/2020.07.15.198010

Figure Lengend Snippet: Heatmap representing average RFUs of HMGB1 binding to sialosides and non-sialosides under various conditions.

Article Snippet: Purified mouse anti-HMGB1 antibody (BioLegend; catalogue number-651402, Lot# B219634) and Cy3-conjugated goat anti-mouse IgG (Jackson ImmunoResearch; catalogue number-115-165-008) were used.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Acidosis, Zinc and HMGB1 in Sepsis: A Common Connection Involving Sialoglycan Recognition

doi: 10.1101/2020.07.15.198010

Figure Lengend Snippet: The binding of HMGB1 with sialoglycan probes on a glycan array was performed using different zinc concentrations. The data shows mean RFU ± SD.

Article Snippet: Purified mouse anti-HMGB1 antibody (BioLegend; catalogue number-651402, Lot# B219634) and Cy3-conjugated goat anti-mouse IgG (Jackson ImmunoResearch; catalogue number-115-165-008) were used.

Techniques: Binding Assay, Glycoproteomics

Journal: bioRxiv

Article Title: Acidosis, Zinc and HMGB1 in Sepsis: A Common Connection Involving Sialoglycan Recognition

doi: 10.1101/2020.07.15.198010

Figure Lengend Snippet: A) The binding of HMGB1 with heparin was determined by ELISA using a binding buffer at different pH ranges (7.1-7.8) B) The binding assay of HMGB1 and heparin was also performed with a binding buffer with and without zinc. The experiment was performed in triplicate, where data shows mean±SD.

Article Snippet: Purified mouse anti-HMGB1 antibody (BioLegend; catalogue number-651402, Lot# B219634) and Cy3-conjugated goat anti-mouse IgG (Jackson ImmunoResearch; catalogue number-115-165-008) were used.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Acidosis, Zinc and HMGB1 in Sepsis: A Common Connection Involving Sialoglycan Recognition

doi: 10.1101/2020.07.15.198010

Figure Lengend Snippet: Different arginine mutants of HMGB1 B-box were generated and binding of these mutants with 3’-sialyllactose was measured by ELISA. The experiment was performed in triplicate, where data shows mean±SD.

Article Snippet: Purified mouse anti-HMGB1 antibody (BioLegend; catalogue number-651402, Lot# B219634) and Cy3-conjugated goat anti-mouse IgG (Jackson ImmunoResearch; catalogue number-115-165-008) were used.

Techniques: Generated, Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Acidosis, Zinc and HMGB1 in Sepsis: A Common Connection Involving Sialoglycan Recognition

doi: 10.1101/2020.07.15.198010

Figure Lengend Snippet: A, B) A schematic showing the binding of HMGB1 to sialic acid under physiological pH and binding to leukocyte receptors at low pH.

Article Snippet: Purified mouse anti-HMGB1 antibody (BioLegend; catalogue number-651402, Lot# B219634) and Cy3-conjugated goat anti-mouse IgG (Jackson ImmunoResearch; catalogue number-115-165-008) were used.

Techniques: Binding Assay

Journal: The Journal of Clinical Investigation

Article Title: Platelet-derived HMGB1 is a critical mediator of thrombosis

doi: 10.1172/JCI81660

Figure Lengend Snippet: (A) Hmgb1 Pf4 mice have prolonged bleeding time compared with Hmgb1 Flox control mice. (B) PT, (C) aPTT, and (D) TT are not altered in Hmgb1 Pf4 mice as compared with control mice. (E and F) Reduction in collagen-induced platelet aggregation in Hmgb1 Pf4 mice. (G) Blood derived from Hmgb1 Pf4 mice is less thrombogenic in a flow chamber system. Exogenous HMGB1 increases thrombus formation in blood from both Hmgb1 Pf4 and Hmgb1 Flox mice. (H–J) FeCl3-induced thrombus formation is inhibited in Hmgb1 Pf4 mice, with (H) improved blood flow and (I) prolonged time to vessel occlusion (as measured by laser Doppler imaging, quantified in J). (K) Immunofluorescence staining of thrombi from Hmgb1 Flox control mice demonstrates large clusters of CD41-positive platelets and surrounding deposition of HMGB1 overlying a fibrinogen network. Thrombi from Hmgb1 Pf4 animals show smaller clusters without HMGB1 expression. Scale bar: 100 μm (top row), 40 μm (bottom row). (L) Ly6G-positive immune cell infiltrates and citH3-positive NETs detected in FeCl3-induced thrombi from Hmgb1 Flox mice but not Hmgb1 Pf4 mice. Scale bar: 100 μm (top row), 40 μm (bottom row). (M) Western blot of clots isolated from Hmgb1 Pf4 mice reveals almost no expression of HMGB1 as compared with control. Data show mean ± SD from at least 3 separate experiments and (B–D) n = 3, (G, H, and J) n = 4, (F) n = 5, and (A) n = 12 mice per group. (E, I, and K–M) Representative images from at least 4 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t test in A–D, F, H, and J; 1-way ANOVA with Tukey’s post-hoc test in G).

Article Snippet: Flow cytometric evaluation of HMGB1 and CD62P expression on platelets Blood samples from trauma patients and healthy volunteers were obtained from the antecubital vein and collected in citrate phosphate dextrose adenine, diluted 1:30 with PBS, and incubated with APC-conjugated anti-CD42b monoclonal antibody (mouse IgG1κ; 17-0429-42, eBioscience) and Alexa Fluor 488–conjugated anti-HMGB1 monoclonal antibody (mouse IgG2B; clone 115603, R&D Systems) or respective isotype control antibodies for 30 minutes.

Techniques: Control, Derivative Assay, Imaging, Immunofluorescence, Staining, Expressing, Western Blot, Isolation

Journal: The Journal of Clinical Investigation

Article Title: Platelet-derived HMGB1 is a critical mediator of thrombosis

doi: 10.1172/JCI81660

Figure Lengend Snippet: (A) Expression of HMGB1 on the surface of circulating platelets is increased in trauma patients as compared with healthy subjects. (B) Systemic aggregation of circulating platelets is upregulated in Hmgb1 Flox mice 30 minutes after induction of experimental trauma/hemorrhagic shock as compared with Hmgb1 Pf4 mice. (C) Small vessel thrombi (H&E staining) and (D) CD41-positive platelet aggregates (immunofluorescence staining and quantification of platelets) are detected in lungs and livers of Hmgb1 Flox mice but not in or only very little in Hmgb1 Pf4 mice after trauma/hemorrhagic shock. Arrows indicate small vessel thrombus formation and vascular congestion. Scale bar: 200 μm (C, top row), 100 μm (C, bottom row, and D, top rows), 50 μm (D, bottom rows). (E) CitH3-positive NETs and Ly6G-positive immune cell infiltrates are decreased in lungs of Hmgb1 Pf4 animals subjected to trauma/hemorrhagic shock compared with Hmgb1 Flox mice subjected to trauma/hemorrhagic shock (immunofluorescence staining and quantification). Scale bar: 10 μm. Data show mean ± SD for (A) n = 4 patients or (B–E) n ≥ 4 mice per group from at least 3 separate experiments in all studies. *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t test in B, D, and E; 1-way ANOVA with Tukey’s post-hoc test in A).

Article Snippet: Flow cytometric evaluation of HMGB1 and CD62P expression on platelets Blood samples from trauma patients and healthy volunteers were obtained from the antecubital vein and collected in citrate phosphate dextrose adenine, diluted 1:30 with PBS, and incubated with APC-conjugated anti-CD42b monoclonal antibody (mouse IgG1κ; 17-0429-42, eBioscience) and Alexa Fluor 488–conjugated anti-HMGB1 monoclonal antibody (mouse IgG2B; clone 115603, R&D Systems) or respective isotype control antibodies for 30 minutes.

Techniques: Expressing, Staining, Immunofluorescence

Journal: The Journal of Clinical Investigation

Article Title: Platelet-derived HMGB1 is a critical mediator of thrombosis

doi: 10.1172/JCI81660

Figure Lengend Snippet: (A) The addition of rHMGB1 to isolated human platelets spreading on collagen or vWF increases final platelet surface area (after 30 minutes), as investigated with SICM at single-cell level. Scale bar: 2 μm. (B) HMGB1 increases the speed of platelet spreading (quantified by area growth exponent) on collagen or vWF within a 9-minute investigation period. Scale bar: 3 μm. Data show mean ± SD for (A) n ≥ 27 or (B) n ≥ 6 from at least 3 separate experiments in all studies. **P < 0.01, ***P < 0.001 (Student’s t test).

Article Snippet: Flow cytometric evaluation of HMGB1 and CD62P expression on platelets Blood samples from trauma patients and healthy volunteers were obtained from the antecubital vein and collected in citrate phosphate dextrose adenine, diluted 1:30 with PBS, and incubated with APC-conjugated anti-CD42b monoclonal antibody (mouse IgG1κ; 17-0429-42, eBioscience) and Alexa Fluor 488–conjugated anti-HMGB1 monoclonal antibody (mouse IgG2B; clone 115603, R&D Systems) or respective isotype control antibodies for 30 minutes.

Techniques: Isolation

Journal: The Journal of Clinical Investigation

Article Title: Platelet-derived HMGB1 is a critical mediator of thrombosis

doi: 10.1172/JCI81660

Figure Lengend Snippet: (A) Tail vein injection of rHMGB1 in C57BL/6 mice results in enhanced clot formation in a FeCl3 model (quantified as time to vessel occlusion). (B) Injection of rHMGB1 in C57BL/6 mice increases platelet sequestration in lungs and livers. Scale bar: 100 μm (top rows), 30 μm (bottom rows). This is quantified in C. (D) In a FeCl3 model, HMGB1 decreases the time to vessel occlusion in Tlr4 Flox control mice; this does not occur in Tlr4 Pf4 mice. (E) HMGB1 treatment of blood from WT mice induces a strong prothrombotic effect, which is reversed when repeated with blood from Tlr4–/– and Myd88–/– mice. Scale bar: 70 μm. (F) HMGB1 treatment of blood from WT mice enhances CRP-induced platelet aggregation; this is reversed when repeated with platelets from Tlr4–/– and Myd88–/– mice. Data show mean ± SD of the results from at least 3 separate experiments and n ≥ 3 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t test in A, C, D, and F; 1-way ANOVA with Tukey’s post-hoc test in E).

Article Snippet: Flow cytometric evaluation of HMGB1 and CD62P expression on platelets Blood samples from trauma patients and healthy volunteers were obtained from the antecubital vein and collected in citrate phosphate dextrose adenine, diluted 1:30 with PBS, and incubated with APC-conjugated anti-CD42b monoclonal antibody (mouse IgG1κ; 17-0429-42, eBioscience) and Alexa Fluor 488–conjugated anti-HMGB1 monoclonal antibody (mouse IgG2B; clone 115603, R&D Systems) or respective isotype control antibodies for 30 minutes.

Techniques: Injection, Control

Journal: The Journal of Clinical Investigation

Article Title: Platelet-derived HMGB1 is a critical mediator of thrombosis

doi: 10.1172/JCI81660

Figure Lengend Snippet: (A) Incubation of WT platelets with rHMGB1 (H) induces activation of cGKI in platelets, as validated by Western blot analysis of VASP phosphorylation (P-VASP). LPS and 8-Br-cGMP were used as positive controls. HMGB1 fails to upregulate VASP phosphorylation in (B) Tlr4–/– and (C) Myd88–/– platelets. (D) The HMGB1-induced prothrombotic effect is reversed in CGKI–/– blood. Scale bar: 70 μm. (E) The HMGB1-induced proaggregatory effect is reversed in CGKI–/– platelets. (F) HMGB1-mediated thrombus formation is inhibited in the presence of the cGKI inhibitor DT-2 (tail vein injections in FeCl3 model), as quantified by time to vessel occlusion. Data show mean ± SD of the results from at least 3 separate experiments and n ≥ 3 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t test in E and F; 1-way ANOVA with Tukey’s post-hoc test in D).

Article Snippet: Flow cytometric evaluation of HMGB1 and CD62P expression on platelets Blood samples from trauma patients and healthy volunteers were obtained from the antecubital vein and collected in citrate phosphate dextrose adenine, diluted 1:30 with PBS, and incubated with APC-conjugated anti-CD42b monoclonal antibody (mouse IgG1κ; 17-0429-42, eBioscience) and Alexa Fluor 488–conjugated anti-HMGB1 monoclonal antibody (mouse IgG2B; clone 115603, R&D Systems) or respective isotype control antibodies for 30 minutes.

Techniques: Incubation, Activation Assay, Western Blot, Phospho-proteomics

Journal: The Journal of Clinical Investigation

Article Title: Platelet-derived HMGB1 is a critical mediator of thrombosis

doi: 10.1172/JCI81660

Figure Lengend Snippet: Incubation of CRP-activated platelets (0.5–2.0 μg/ml) with (A) rHMGB1 or (B) LPS enhances the expression of CD62P on the platelet surface, as evaluated by flow cytometry. (C and D) HMGB1-induced enhanced expression of CD62P on CRP-activated WT platelets does not occur on Tlr4–/–, Myd88–/–, or CGKI–/– platelets. (E) Collagen-induced CD62P expression on the surface of HMGB1-deficient platelets is reduced, as compared with Flox control platelets. The addition of rHMGB1 enhances CD62P expression on platelets derived both from Hmgb1 Pf4 mice and Flox control mice, with stronger effects in the latter. (F and G) Incubation of CRP-activated WT platelets with rHMGB1 enhances ATP release, which does not occur in Tlr4–/–, Myd88–/–, or CGKI–/– platelets. Data show mean ± SD of the results from at least 3 separate experiments and n ≥ 4 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t test in A and B; 1-way ANOVA with Tukey’s post-hoc test in C–G).

Article Snippet: Flow cytometric evaluation of HMGB1 and CD62P expression on platelets Blood samples from trauma patients and healthy volunteers were obtained from the antecubital vein and collected in citrate phosphate dextrose adenine, diluted 1:30 with PBS, and incubated with APC-conjugated anti-CD42b monoclonal antibody (mouse IgG1κ; 17-0429-42, eBioscience) and Alexa Fluor 488–conjugated anti-HMGB1 monoclonal antibody (mouse IgG2B; clone 115603, R&D Systems) or respective isotype control antibodies for 30 minutes.

Techniques: Incubation, Expressing, Flow Cytometry, Control, Derivative Assay

Journal: The Journal of Clinical Investigation

Article Title: Platelet-derived HMGB1 is a critical mediator of thrombosis

doi: 10.1172/JCI81660

Figure Lengend Snippet: Detection of intracellular (A) MyD88 and (A and B) GC in isolated (A) human and (B) murine (WT, Myd88–/–) platelets by immunofluorescence staining and confocal laser scanning microscopy. (A) Treatment of human platelets with HMGB1 or LPS induces translocation of MyD88 and GC toward the plasma membrane in platelets, whereas untreated and DEA/NO-treated platelets show a uniform intracellular distribution of MyD88 and GC. Scale bar: 5 μm (top 3 rows), 1 μm (bottom row). (B) HMGB1 also induces translocation of GC toward the plasma membrane in murine WT platelets, which does not occur in Myd88–/– platelets. Scale bar: 5 μm (top row), 1 μm (bottom row). (C–F) Coimmunoprecipitation studies reveal HMGB1-dependent complex formation of MyD88 and GC in murine platelets. (G) HMGB1 treatment induces cGMP production in WT platelets, which does not occur in Myd88–/– platelets. Data show mean ± SD of the results from at least 3 separate experiments and n ≥ 3 mice per group.

Article Snippet: Flow cytometric evaluation of HMGB1 and CD62P expression on platelets Blood samples from trauma patients and healthy volunteers were obtained from the antecubital vein and collected in citrate phosphate dextrose adenine, diluted 1:30 with PBS, and incubated with APC-conjugated anti-CD42b monoclonal antibody (mouse IgG1κ; 17-0429-42, eBioscience) and Alexa Fluor 488–conjugated anti-HMGB1 monoclonal antibody (mouse IgG2B; clone 115603, R&D Systems) or respective isotype control antibodies for 30 minutes.

Techniques: Isolation, Immunofluorescence, Staining, Confocal Laser Scanning Microscopy, Translocation Assay, Clinical Proteomics, Membrane

Journal: The Journal of Clinical Investigation

Article Title: Platelet-derived HMGB1 is a critical mediator of thrombosis

doi: 10.1172/JCI81660

Figure Lengend Snippet: Platelet-derived HMGB1 promotes platelet activation, aggregation, and thrombus formation via TLR4- and MyD88-dependent recruitment of GC toward the platelet plasma membrane, followed by MyD88/GC complex formation, GC activation, and cGMP-dependent activation of cGKI in platelets. sGC, soluble GC.

Article Snippet: Flow cytometric evaluation of HMGB1 and CD62P expression on platelets Blood samples from trauma patients and healthy volunteers were obtained from the antecubital vein and collected in citrate phosphate dextrose adenine, diluted 1:30 with PBS, and incubated with APC-conjugated anti-CD42b monoclonal antibody (mouse IgG1κ; 17-0429-42, eBioscience) and Alexa Fluor 488–conjugated anti-HMGB1 monoclonal antibody (mouse IgG2B; clone 115603, R&D Systems) or respective isotype control antibodies for 30 minutes.

Techniques: Derivative Assay, Activation Assay, Clinical Proteomics, Membrane

Journal: Mediators of Inflammation

Article Title: Elevation of High-Mobility Group Protein Box-1 in Serum Correlates with Severity of Acute Intracerebral Hemorrhage

doi: 10.1155/2010/142458

Figure Lengend Snippet: Serum level of HMGB1 increased significantly in patients with acute ICH. Data are presented as mean ± standard deviation. (a) ELISA assay of the changes of HMGB1 serum levels in patients. (b) A representative picture of Western blot assay showing the changes of HMGB1 serum levels in patients. GAPDH was chosen as the internal control. The changes of HMGB1 were expressed as the ratio of the optical density values of HMGB1 band to the optical density values of GAPDH band. **versus control; P < .01; ## versus good outcome group; P < .01.

Article Snippet: The ECL membranes were incubated with the primary antibodies including mouse antihuman HMGB1 (for HMGB1 assay in serum, 1 : 1000, Santa Cruz) or rabbit antimouse HMGB1 (for HMGB1 assay in brain tissue, 1 : 5000, Santa Cruz), followed by incubation with peroxidase-conjugated secondary antibodies (1 : 2000, Jingmei, China).

Techniques: Standard Deviation, Enzyme-linked Immunosorbent Assay, Western Blot, Control

Journal: Mediators of Inflammation

Article Title: Elevation of High-Mobility Group Protein Box-1 in Serum Correlates with Severity of Acute Intracerebral Hemorrhage

doi: 10.1155/2010/142458

Figure Lengend Snippet: The level of HMGB1 increased significantly in the perihematomal tissue of ICH mice. Data are expressed as mean ± standard deviation. (a) ELISA assay of the changes of HMGB1 levels in the perihematomal tissue of ICH mice. (b) A representative picture of Western blot assay showing the changes of HMGB1 levels in the perihematomal tissue of mice. GAPDH was chosen as the internal control. The changes of HMGB1 were expressed as the ratio of the optical density values of HMGB1 band to the optical density values of GAPDH band. n = 15. **versus control; P < .01; ## versus 72 hours; P < .01.

Article Snippet: The ECL membranes were incubated with the primary antibodies including mouse antihuman HMGB1 (for HMGB1 assay in serum, 1 : 1000, Santa Cruz) or rabbit antimouse HMGB1 (for HMGB1 assay in brain tissue, 1 : 5000, Santa Cruz), followed by incubation with peroxidase-conjugated secondary antibodies (1 : 2000, Jingmei, China).

Techniques: Standard Deviation, Enzyme-linked Immunosorbent Assay, Western Blot, Control

Journal: Mediators of Inflammation

Article Title: Elevation of High-Mobility Group Protein Box-1 in Serum Correlates with Severity of Acute Intracerebral Hemorrhage

doi: 10.1155/2010/142458

Figure Lengend Snippet: Heme could stimulate the secretion of HMGB1 by cultured microglia. Data are expressed as mean ± standard deviation, n = 6. (a) Microglia were cultured with various concentrations of heme (0 μ M, 10 μ M, 20 μ M, and 30 μ M) for 24 hours. Thereafter, the culture medium was replaced, and the cells were further cultured for 12 hours before collection of the supernatant for ELISA determination of HMGB1 levels. **versus 0 μ M; P < .01; ## versus 10 μ M, 20 μ M; P < .01. (b) Microglia were treated with 30 μ M of heme for various lengths of time (0 h, 4 h, 8 h, 24 h, and 48 h). Thereafter, the culture medium was replaced, and the cells were further cultured for another 12 hours before collection of the supernatant for ELISA determination of HMGB1 levels. **versus 0 h; P < .01; ## versus 4 h, 8 h, or 24 h; P < .01. (c) Microglia were treated with 30 μ M of heme, 30 μ M of FeCl 3 , or 30 μ M of FeSO 4 for 24 hours. Thereafter, the culture medium was replaced, and the cells were further cultured for another 12 hours before collection of the supernatant for ELISA determination of HMGB1 levels. **versus control; P < .01.

Article Snippet: The ECL membranes were incubated with the primary antibodies including mouse antihuman HMGB1 (for HMGB1 assay in serum, 1 : 1000, Santa Cruz) or rabbit antimouse HMGB1 (for HMGB1 assay in brain tissue, 1 : 5000, Santa Cruz), followed by incubation with peroxidase-conjugated secondary antibodies (1 : 2000, Jingmei, China).

Techniques: Cell Culture, Standard Deviation, Enzyme-linked Immunosorbent Assay, Control

Journal: Experimental neurology

Article Title: High-Mobility Group Box-1 Translocation and Release after Hypoxic Ischemic Brain Injury in Neonatal Rats

doi: 10.1016/j.expneurol.2018.09.007

Figure Lengend Snippet: (A) Representative images of neonatal rat brains obtained from sham operated and zero, 3 6, 12, 24 and 48 h after 2 h of hypoxia and carotid artery ligation. A sham operated neonatal rat (P7) shows nuclear HMGB1 staining in most cells (white arrows) of the cerebral cortical region. Immediately at 0 h after HI, HMGB1 exhibited translocation from the cellular nuclei into the cytosol (0, 3, and 12 h, yellow arrows) in the cerebral cortex of the ipsilateral HI brain hemisphere. At 6 24, and 48 h after HI, HMGB1 exhibited punctate structures with strong signals in extracellular matrix and along neuronal cell somata and axons (6, 12, 24, and 48 h, thick white arrows). A schematic illustration showing the brain region (highlighted in gray) that exhibited HMGB1 translocation and release (right corner). All images are from the HI region of the cerebral cortex. Scale bar = 50 μm. (B) Quantification of total HMGB1 cytoplasmic translocation in the cerebral cortical cells of sham-operated, zero, 3, 12, and 48h after HI in the neonatal rats. HMGB1 cytoplasmic translocation was increased at zero, 3, and 12 h after exposure to HI compared to the sham group. Mean ± SD, n= 4 for each group. *P<0.05 versus Sham, ANOVA, Fisher LSD.

Article Snippet: The sections were then incubated overnight with a primary antibody solution with mouse anti-HMGB1 monoclonal antibody (R&D Systems Inc., Minneapolis, MN, USA) at a dilution of 1:500 in combination with either a rabbit anti-NeuN polyclonal antibody (Abcam, Cambridge, MA, USA) at a dilution of 1:1000, or rabbit anti-Iba-1 monoclonal antibody (Dako, Carpinteria, CA, USA) at a dilution of 1:1000, or a rabbit anti-GFAP polyclonal antibody (Dako, Carpinteria, CA, USA) at a dilution of 1:1000.

Techniques: Ligation, Staining, Translocation Assay

Journal: Experimental neurology

Article Title: High-Mobility Group Box-1 Translocation and Release after Hypoxic Ischemic Brain Injury in Neonatal Rats

doi: 10.1016/j.expneurol.2018.09.007

Figure Lengend Snippet: HMGB1 translocation was observed as early as zero and 3 h after HI injury (zero and 3 h, yellow arrows). HMGB1 release was detected at 6 h after HI injury (6 h, thick white arrows). Major release of HMGB1 into extracellular compartment was observed at 48 h after HI injury (48 h, thick white arrows). Scale bar = 20 μm.

Article Snippet: The sections were then incubated overnight with a primary antibody solution with mouse anti-HMGB1 monoclonal antibody (R&D Systems Inc., Minneapolis, MN, USA) at a dilution of 1:500 in combination with either a rabbit anti-NeuN polyclonal antibody (Abcam, Cambridge, MA, USA) at a dilution of 1:1000, or rabbit anti-Iba-1 monoclonal antibody (Dako, Carpinteria, CA, USA) at a dilution of 1:1000, or a rabbit anti-GFAP polyclonal antibody (Dako, Carpinteria, CA, USA) at a dilution of 1:1000.

Techniques: Translocation Assay

Journal: Experimental neurology

Article Title: High-Mobility Group Box-1 Translocation and Release after Hypoxic Ischemic Brain Injury in Neonatal Rats

doi: 10.1016/j.expneurol.2018.09.007

Figure Lengend Snippet: (A) The percent of nuclear staining of HMGB1 and NeuN positive cells to total DAPI positive cells decreased at 3, 12, and 48 h after HI injury. (B) The percent of cytoplasmic double staining of HMGB1 and NeuN positive cells to total DAPI positive cells increased at 3 and 12 h, but decreased at 48 h after HI injury. (C) The percent of HMGB1 negative and NeuN positive cells to total DAPI stained cells increased at 12 and 48 h after HI injury. Mean ± SD, n = 4 for each group except n = 3 for the 12 h group. * P < 0.05 versus Sham, ANOVA, Fisher LSD.

Article Snippet: The sections were then incubated overnight with a primary antibody solution with mouse anti-HMGB1 monoclonal antibody (R&D Systems Inc., Minneapolis, MN, USA) at a dilution of 1:500 in combination with either a rabbit anti-NeuN polyclonal antibody (Abcam, Cambridge, MA, USA) at a dilution of 1:1000, or rabbit anti-Iba-1 monoclonal antibody (Dako, Carpinteria, CA, USA) at a dilution of 1:1000, or a rabbit anti-GFAP polyclonal antibody (Dako, Carpinteria, CA, USA) at a dilution of 1:1000.

Techniques: Staining, Double Staining

Journal: Experimental neurology

Article Title: High-Mobility Group Box-1 Translocation and Release after Hypoxic Ischemic Brain Injury in Neonatal Rats

doi: 10.1016/j.expneurol.2018.09.007

Figure Lengend Snippet: The HMGB1 expression was mainly in the nucleus of astrocytes in the sham-operated, and at zero, 3, 6, 12, and 24 h after HI. HMGB1 translocation and release was not observed in the astrocytes. DNA damage with ring-like structures co-localized with HMGB1 was observed in some astrocytes (3 h, white arrowhead). Furthermore, some astrocytes showed negative nuclear staining of HMGB1 at 48 h after HI injury (48 h, white arrowhead). White arrows show the representative nuclear localization of HMGB1 in GFAP positive cells. Scale bar = 20 μm.

Article Snippet: The sections were then incubated overnight with a primary antibody solution with mouse anti-HMGB1 monoclonal antibody (R&D Systems Inc., Minneapolis, MN, USA) at a dilution of 1:500 in combination with either a rabbit anti-NeuN polyclonal antibody (Abcam, Cambridge, MA, USA) at a dilution of 1:1000, or rabbit anti-Iba-1 monoclonal antibody (Dako, Carpinteria, CA, USA) at a dilution of 1:1000, or a rabbit anti-GFAP polyclonal antibody (Dako, Carpinteria, CA, USA) at a dilution of 1:1000.

Techniques: Expressing, Translocation Assay, Staining

Journal: Experimental neurology

Article Title: High-Mobility Group Box-1 Translocation and Release after Hypoxic Ischemic Brain Injury in Neonatal Rats

doi: 10.1016/j.expneurol.2018.09.007

Figure Lengend Snippet: HMGB1 translocation and release in GFAP positive cells after HI injury in neonatal rats.

Article Snippet: The sections were then incubated overnight with a primary antibody solution with mouse anti-HMGB1 monoclonal antibody (R&D Systems Inc., Minneapolis, MN, USA) at a dilution of 1:500 in combination with either a rabbit anti-NeuN polyclonal antibody (Abcam, Cambridge, MA, USA) at a dilution of 1:1000, or rabbit anti-Iba-1 monoclonal antibody (Dako, Carpinteria, CA, USA) at a dilution of 1:1000, or a rabbit anti-GFAP polyclonal antibody (Dako, Carpinteria, CA, USA) at a dilution of 1:1000.

Techniques: Translocation Assay, Staining, Negative Staining

Journal: Experimental neurology

Article Title: High-Mobility Group Box-1 Translocation and Release after Hypoxic Ischemic Brain Injury in Neonatal Rats

doi: 10.1016/j.expneurol.2018.09.007

Figure Lengend Snippet: The HMGB1 expression was detected in the nucleus of the microglial cells in the sham-operated, and in the zero, 3, 6, 12, and 24 h after HI. HMGB1 translocation and release were not observed in microglia. White arrows show the representative nuclear localization of HMGB1 in the Iba-1 positive cells. Scale bar = 20 μm.

Article Snippet: The sections were then incubated overnight with a primary antibody solution with mouse anti-HMGB1 monoclonal antibody (R&D Systems Inc., Minneapolis, MN, USA) at a dilution of 1:500 in combination with either a rabbit anti-NeuN polyclonal antibody (Abcam, Cambridge, MA, USA) at a dilution of 1:1000, or rabbit anti-Iba-1 monoclonal antibody (Dako, Carpinteria, CA, USA) at a dilution of 1:1000, or a rabbit anti-GFAP polyclonal antibody (Dako, Carpinteria, CA, USA) at a dilution of 1:1000.

Techniques: Expressing, Translocation Assay

Journal: Experimental neurology

Article Title: High-Mobility Group Box-1 Translocation and Release after Hypoxic Ischemic Brain Injury in Neonatal Rats

doi: 10.1016/j.expneurol.2018.09.007

Figure Lengend Snippet: (A) Double immunohistochemical staining of HMGB1 and ApopTag 48 h after exposure to HI: ApopTag+/HMGB1− cells shown white arrows; ApopTag− /HMGB1− cells shown by thick white arrows. Scale bar = 50 μm. (B) Quantification of apoptotic cells (ApopTag+ in Sham and 48 h after HI, blue bar), apoptotic positive cells with depleted HMGB1 (ApopTag+/HMGB1−, red bar), and non-apoptotic cells with depleted HMGB1 (ApopTag−/HMGB1−, green bar) after HI injury. Bar graphs represent the percent of apoptotic cells, which have released HMGB1 48 h after HI injury. Mean ± SD, n = 6, three cerebral cortical regions counted for each rat brain. *P<0.001 by ANOVA, Fisher LSD. (C) (a) 2,3,5-triphenyltetrazolium chloride (TTC) staining of brain section. Dashed line shows HI region examined. (b) Double immunohistochemical staining for HMGB1 and LC3-II as an autophagic cellular marker. HMGB1 translocation is co-localized with the autophagic cells in a circumscribed region of the cerebral cortex; Scale bar =10 μm.

Article Snippet: The sections were then incubated overnight with a primary antibody solution with mouse anti-HMGB1 monoclonal antibody (R&D Systems Inc., Minneapolis, MN, USA) at a dilution of 1:500 in combination with either a rabbit anti-NeuN polyclonal antibody (Abcam, Cambridge, MA, USA) at a dilution of 1:1000, or rabbit anti-Iba-1 monoclonal antibody (Dako, Carpinteria, CA, USA) at a dilution of 1:1000, or a rabbit anti-GFAP polyclonal antibody (Dako, Carpinteria, CA, USA) at a dilution of 1:1000.

Techniques: Immunohistochemical staining, Staining, Marker, Translocation Assay

Journal: Experimental neurology

Article Title: High-Mobility Group Box-1 Translocation and Release after Hypoxic Ischemic Brain Injury in Neonatal Rats

doi: 10.1016/j.expneurol.2018.09.007

Figure Lengend Snippet: (A) Serum collected from sham operated (n=17) and HI rats immediately 0 h (n=5), 3 h (n=12), 6 h (n=14), 12 h (n=12), 24 h (n=11), and 48 h (n=15) h after exposure to HI. Mean ± SD, *P<0.05, ANOVA, Fisher LSD. (B) Representative Western immunoblots for β-Actin (43KD) and HMGB1 (28 kD) shown for the internal control protein (IC), in Sham control, and 3, 12 and 48 h after HI. Total protein extracted from ipsilateral-HI cerebral cortical hemisphere from rat brain of sham treated, 3, 12, and 48 h after exposure to HI (each group: n=5). Total HMGB1 expression in rat brain, plotted as ratio to internal control (IC). Mean ± SD, * P<0.05, ANOVA, Fisher LSD.

Article Snippet: The sections were then incubated overnight with a primary antibody solution with mouse anti-HMGB1 monoclonal antibody (R&D Systems Inc., Minneapolis, MN, USA) at a dilution of 1:500 in combination with either a rabbit anti-NeuN polyclonal antibody (Abcam, Cambridge, MA, USA) at a dilution of 1:1000, or rabbit anti-Iba-1 monoclonal antibody (Dako, Carpinteria, CA, USA) at a dilution of 1:1000, or a rabbit anti-GFAP polyclonal antibody (Dako, Carpinteria, CA, USA) at a dilution of 1:1000.

Techniques: Western Blot, Control, Expressing

Journal: Experimental neurology

Article Title: High-Mobility Group Box-1 Translocation and Release after Hypoxic Ischemic Brain Injury in Neonatal Rats

doi: 10.1016/j.expneurol.2018.09.007

Figure Lengend Snippet: (A) Representative images of HMGB1 double immunochistochemical staining in sham treated and 12 and 48 h after hypoxia alone. Double immunohistochemical staining of HMGB1 and the neuronal marker (NeuN) from rat cerebral cortex 48 h after hypoxia. (B) Quantification of the percent of cells exhibiting HMBG1 translocation from the nucleus to the cytosolic compartment in after 2 h of hypoxia alone. Mean ± SD, n=4 for each group except n=3 for the 48 h group. *P<0.05 versus Sham, ANOVA, Fisher LSD.

Article Snippet: The sections were then incubated overnight with a primary antibody solution with mouse anti-HMGB1 monoclonal antibody (R&D Systems Inc., Minneapolis, MN, USA) at a dilution of 1:500 in combination with either a rabbit anti-NeuN polyclonal antibody (Abcam, Cambridge, MA, USA) at a dilution of 1:1000, or rabbit anti-Iba-1 monoclonal antibody (Dako, Carpinteria, CA, USA) at a dilution of 1:1000, or a rabbit anti-GFAP polyclonal antibody (Dako, Carpinteria, CA, USA) at a dilution of 1:1000.

Techniques: Staining, Immunohistochemical staining, Marker, Translocation Assay

Journal: Experimental neurology

Article Title: High-Mobility Group Box-1 Translocation and Release after Hypoxic Ischemic Brain Injury in Neonatal Rats

doi: 10.1016/j.expneurol.2018.09.007

Figure Lengend Snippet: HMGB1 cytoplasmic translocation was significantly higher at zero, 3, and 12 h in Hi-treated group (closed bar) than in hypoxia alone group (gray bar). Due to HMGB1 release, HMGB1 cytoplasmic translocation was lower at 48 h in HI treated group than in hypoxia alone group. Mean ± SD, n=4 for each group except n=3 for the 48 h hypoxia group. *P<0.05, two-way ANOVA, Fisher LSD.

Article Snippet: The sections were then incubated overnight with a primary antibody solution with mouse anti-HMGB1 monoclonal antibody (R&D Systems Inc., Minneapolis, MN, USA) at a dilution of 1:500 in combination with either a rabbit anti-NeuN polyclonal antibody (Abcam, Cambridge, MA, USA) at a dilution of 1:1000, or rabbit anti-Iba-1 monoclonal antibody (Dako, Carpinteria, CA, USA) at a dilution of 1:1000, or a rabbit anti-GFAP polyclonal antibody (Dako, Carpinteria, CA, USA) at a dilution of 1:1000.

Techniques: Translocation Assay