Journal: The Journal of Biological Chemistry

Article Title: Activated Platelets Interfere with Recruitment of Mesenchymal Stem Cells to Apoptotic Cardiac Cells via High Mobility Group Box 1/Toll-like Receptor 4-mediated Down-regulation of Hepatocyte Growth Factor Receptor MET *

doi: 10.1074/jbc.M113.530287

Figure Lengend Snippet: Expression of HMGB1 in platelets and release upon platelet activation. A, expression of intracellular HMGB1 in resting (untreated) and activated (5 μg/ml CRP, 15 min; 50 μm ADP, 15 min) platelets was determined by immunofluorescence staining and confocal laser-scanning microscopy. An HMGB1-specific polyclonal antibody and an Alexa Fluor 488-tagged antibody (green) were used. Representative images of three independent experiments. Scale bar, 5 μm. B, HMGB1 protein levels in lysates and conditioned media derived from resting and CRP-activated platelets (5 μg/ml CRP; 5, 15, and 30 min) were deciphered by Western blot analysis, detecting HMGB1 at 25 kDa. An actin polyclonal antibody served as loading control. Statistical significance (*, p < 0.04; **, p < 0.01) of densitometric analysis of HMGB1 bands (normalized to actin) is indicated. C, HMGB1 levels in conditioned media derived from resting (untreated, 5, 15, 30 min) and activated (50 μm ADP, 5, 15, 30 min; 5 μg/ml CRP, 5, 15, 30 min) platelets were also measured by ELISA. In addition, surface expression of P-selectin (CD62P) on platelets was investigated by staining with a CD62P-specific monoclonal antibody and flow cytometry. Error bars, S.E.

Article Snippet: Cells were incubated overnight at 4 °C with anti-HGF (C-terminal) polyclonal antibody (5 μg/ml; rabbit Ig; Abgent, San Diego, CA), washed with PBS + 0.3% Triton X-100 + 0.1% Tween 20, and incubated with Alexa Fluor 488-tagged goat anti-rabbit IgG (1:100) for 2 h at room temperature.

Techniques: Expressing, Activation Assay, Immunofluorescence, Staining, Confocal Laser Scanning Microscopy, Derivative Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: The Journal of Biological Chemistry

Article Title: Activated Platelets Interfere with Recruitment of Mesenchymal Stem Cells to Apoptotic Cardiac Cells via High Mobility Group Box 1/Toll-like Receptor 4-mediated Down-regulation of Hepatocyte Growth Factor Receptor MET *

doi: 10.1074/jbc.M113.530287

Figure Lengend Snippet: Contribution of individual HMGB1 receptors to the platelet-mediated inhibition of MSC migration to apoptotic cardiac myocytes and fibroblasts. A, expression of HMGB1 receptors RAGE, TLR-2, and TLR-4 on MSC was determined by flow cytometry after staining with specific antibodies. Histograms were set according to negative control stainings (#). MSC were preincubated with conditioned media derived from CRP-activated (5 μg/ml CRP, 30 min) (B and D) or ADP-activated (50 μm ADP, 30 min) platelets (C and E) in the presence or absence of neutralizing antibodies to RAGE, TLR-2, and TLR-4 before migration toward conditioned media of staurosporine-treated or sodium azide-treated cardiac myocytes (B and C) or cardiac fibroblasts (D and E) was assessed in transwell migration experiments. F, knockdown of TLR-4 expression in MSC was carried out through transfection of MSC with siRNA against TLR-4 and validated by Western blot analysis, detecting TLR-4 at 95 kDa. A non-targeting sequence and untreated MSC served as controls for transfection; an actin polyclonal antibody served as loading control. Statistical significance (*, p < 0.05) of densitometric analysis of TLR-4 bands (normalized to actin) is indicated. To evaluate the migratory potential of transfected or non-transfected MSC, cells were incubated with conditioned media derived from CRP-activated (5 μg/ml CRP, 30 min) or ADP-activated (50 μm ADP, 30 min) platelets before migration toward conditioned media of staurosporine-treated or sodium azide-treated cardiac myocytes (G) or cardiac fibroblasts (H) was assessed in transwell migration experiments. Cell migration data (mean ± S.E. (error bars) for n ≥ 3) are presented as percentage inhibition calculated from the number of migrating MSC that have not been preincubated with conditioned media derived from activated platelets. Statistical significance (*, p < 0.05; **, p < 0.007) of cell migration data is indicated.

Article Snippet: Cells were incubated overnight at 4 °C with anti-HGF (C-terminal) polyclonal antibody (5 μg/ml; rabbit Ig; Abgent, San Diego, CA), washed with PBS + 0.3% Triton X-100 + 0.1% Tween 20, and incubated with Alexa Fluor 488-tagged goat anti-rabbit IgG (1:100) for 2 h at room temperature.

Techniques: Inhibition, Migration, Expressing, Flow Cytometry, Staining, Negative Control, Derivative Assay, Transfection, Western Blot, Sequencing, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Activated Platelets Interfere with Recruitment of Mesenchymal Stem Cells to Apoptotic Cardiac Cells via High Mobility Group Box 1/Toll-like Receptor 4-mediated Down-regulation of Hepatocyte Growth Factor Receptor MET *

doi: 10.1074/jbc.M113.530287

Figure Lengend Snippet: Role of HGF in apoptosis-induced migration of MSC. A, expression of intracellular HGF in vital (untreated), apoptotic (300 nm staurosporine, 3 h), and necrotic (40 μm H2O2, 3 h) cardiac myocytes was evaluated by immunofluorescence staining and confocal laser-scanning microscopy. An HGF-specific polyclonal antibody and an Alexa Fluor 488-tagged antibody (green) were used. Blue, nuclear staining with TO-PRO-3 iodide. Representative images of two independent experiments are shown. Scale bar, 20 μm. B, HGF levels in conditioned media derived from vital (untreated), apoptotic (300 nm staurosporine, 3 h; 10 mm sodium azide, 3 h), or necrotic (40 μm H2O2, 3 h; 25% ethanol, 1 h) cardiac myocytes or cardiac fibroblasts were measured by ELISA. Conditioned media derived from staurosporine-treated or sodium azide-treated cardiac myocytes or cardiac fibroblasts in the presence or absence of 1 μg/ml anti-HGF neutralizing antibody or control antibody (C) or graded doses of recombinant HGF (D) served as targets in transwell migration experiments. Data are presented as mean ± S.E. (error bars) (n ≥ 3). Statistical significance (**, p ≤ 0.002) is indicated.

Article Snippet: Cells were incubated overnight at 4 °C with anti-HGF (C-terminal) polyclonal antibody (5 μg/ml; rabbit Ig; Abgent, San Diego, CA), washed with PBS + 0.3% Triton X-100 + 0.1% Tween 20, and incubated with Alexa Fluor 488-tagged goat anti-rabbit IgG (1:100) for 2 h at room temperature.

Techniques: Migration, Expressing, Immunofluorescence, Staining, Confocal Laser Scanning Microscopy, Derivative Assay, Enzyme-linked Immunosorbent Assay, Recombinant

Journal: The Journal of Biological Chemistry

Article Title: Activated Platelets Interfere with Recruitment of Mesenchymal Stem Cells to Apoptotic Cardiac Cells via High Mobility Group Box 1/Toll-like Receptor 4-mediated Down-regulation of Hepatocyte Growth Factor Receptor MET *

doi: 10.1074/jbc.M113.530287

Figure Lengend Snippet: Effect of platelet activation on expression of HGF receptor MET on MSC. A, MSC were preincubated with graded doses of recombinant HMGB1 (10–40 ng/ml) for 24 h and then tested for migration toward recombinant HGF (20 ng/ml) in the presence or absence of a neutralizing TLR-4 antibody. The number of migrating MSC was determined after 12 h. B, surface expression of HGF receptor MET on untreated MSC and MSC incubated with conditioned media derived from ADP- or CRP-activated platelets for 24 h was evaluated by immunofluorescence staining and confocal laser-scanning microscopy. A MET-specific polyclonal antibody and an Alexa Fluor 488-tagged antibody (green) were used. Blue, nuclear staining with TO-PRO-3 iodide. Shown is quantification of MET expression by mean fluorescence intensity. Statistical significance (*, p < 0.02) is indicated. Representative images of three independent experiments are shown. Scale bar, 40 μm. C and D, expression of MET on untreated MSC and MSC incubated with conditioned media derived from ADP- or CRP-activated platelets (2, 4, 12, and 24 h) in the presence or absence of neutralizing antibodies to HMGB1 or TLR-4 was also determined by flow cytometry and quantified by mean fluorescence intensity. E, down-regulation of MET on MSC was further studied by fractionation of untreated MSC and MSC incubated with conditioned media derived from ADP- or CRP-activated platelets for 12 h into plasma membrane and cytosolic components, and subsequent Western blot analysis was performed on the distinct fractions, detecting MET at 145 and 125 kDa. Data are presented as mean ± S.E. (error bars) (n ≥ 3). Statistical significance (*, p < 0.05) of densitometric analysis of MET bands (normalized to actin) is indicated.

Article Snippet: Cells were incubated overnight at 4 °C with anti-HGF (C-terminal) polyclonal antibody (5 μg/ml; rabbit Ig; Abgent, San Diego, CA), washed with PBS + 0.3% Triton X-100 + 0.1% Tween 20, and incubated with Alexa Fluor 488-tagged goat anti-rabbit IgG (1:100) for 2 h at room temperature.

Techniques: Activation Assay, Expressing, Recombinant, Migration, Incubation, Derivative Assay, Immunofluorescence, Staining, Confocal Laser Scanning Microscopy, Fluorescence, Flow Cytometry, Fractionation, Western Blot

Journal: Molecular Cancer Therapeutics

Article Title: Preclinical Profile of BYON3521 Predicts an Effective and Safe MET Antibody–Drug Conjugate

doi: 10.1158/1535-7163.MCT-22-0596

Figure Lengend Snippet: Competition experiments with rhHGF and MET ECD. A, BLI experiment to assess competition of rhHGF for MET ECD binding of SYD2884. Presented are the binding curve of SYD2884 visualized with an HRP-conjugated anti-hIgG (red line, solid squares) and rhHGF visualized using a polyclonal anti-HGF antibody (blue line, solid triangles). The detectable amount of SYD2884 binding to the recombinant human MET ECD, immobilized on the surface of the sensor tip, decreases whereas at the same time the detectable amount of rhHGF increases. The data are presented as the correlation between the measured signals versus the molar excess of fully activated rhHGF (left graph) and pro-HGF (right graph). B, Cell viability of EBC-1 cells treated with increasing concentrations of BYON3521 or nonbinding isotype control ADC in the presence or absence of 1 μg/mL MET ECD, either administered together directly, or after 30-minute preincubation of BYON3521 or isotype control ADC and MET ECD (Premix). (Approximate MW of BYON3521 is 149 kD, of SY2884 144 kD, and MET ECD 140 kD).

Article Snippet: SYD2884 binding was monitored using an anti-hIgG-HRP (The Binding Site, Birmingham, United Kingdom; AP003.M, 5 minutes, 1:500), HGF binding was detected using a polyclonal anti-HGF antibody (Abnova, Taipei City, Taiwan; H0003082-DOIP, 10 minutes, 20 μg/mL).

Techniques: Binding Assay, Recombinant, Control