Journal: Frontiers in Microbiology

Article Title: A microsatellite DNA-derived oligodeoxynucleotide attenuates lipopolysaccharide-induced acute lung injury in mice by inhibiting the HMGB1-TLR4-NF-κB signaling pathway

doi: 10.3389/fmicb.2022.964112

Figure Lengend Snippet: Suppression of HMGB1 translocation and secretion in RAW264.7 cells treated with MS19 after LPS exposure. Immunofluorescence images of nucleocytoplasmic translocation of HMGB1 (A) . RAW264.7 cells were incubated with LPS with or without MS19 for 2 h and examined by fluorescence microscopy. HMGB1 are stained in green with FITC, and cell nuclei are stained in blue with DAPI. The percentage of nucleocytoplasmic translocation of HMGB1 in RAW264.7 cells was calculated (B) . The HMGB1 level in supernatant after RAW264.7 cells received 16 h exposure of LPS in the presence or absence of MS19, as determined using ELISA (C) . Data are representatives of three independent experiments and are expressed as means ± SEM. *** p < 0.001, vs. medium group; ## p < 0.01, vs. LPS group.

Article Snippet: After washing with PBS, cells were incubated with goat anti-rabbit IgG against HMGB1 conjugated with FITC (1:200; Beyotime) for 1 h at room temperature.

Techniques: Translocation Assay, Immunofluorescence, Incubation, Fluorescence, Microscopy, Staining, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Microbiology

Article Title: A microsatellite DNA-derived oligodeoxynucleotide attenuates lipopolysaccharide-induced acute lung injury in mice by inhibiting the HMGB1-TLR4-NF-κB signaling pathway

doi: 10.3389/fmicb.2022.964112

Figure Lengend Snippet: Protective effect of MS19 on LPS-induced acute lung injury in mice. Mice ( n = 11) were challenged nasally with LPS or PBS at 6 h after intravenous injection with MS19 or PBS. The body weight change of mice (A) . The percent survival of mice in different groups at 8 days after LPS challenge (B) . Representative images of lung pathology after H&E staining (C) . Lung injury scores (D) . ELISA of HMGB1, IL-6, and TNF-α level in the BALF samples of mice in each group (E) . Data represent the mean ± SEM. * p < 0.05, ** p < 0.01, and *** p < 0.001, vs. PBS group; # p < 0.05, ## p < 0.01, and ### p < 0.001, vs. LPS group.

Article Snippet: After washing with PBS, cells were incubated with goat anti-rabbit IgG against HMGB1 conjugated with FITC (1:200; Beyotime) for 1 h at room temperature.

Techniques: Injection, Staining, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Microbiology

Article Title: A microsatellite DNA-derived oligodeoxynucleotide attenuates lipopolysaccharide-induced acute lung injury in mice by inhibiting the HMGB1-TLR4-NF-κB signaling pathway

doi: 10.3389/fmicb.2022.964112

Figure Lengend Snippet: Scheme of the anti-inflammatory mechanisms of MS19 via the inhibition of NF-κB signaling and HMGB1 release in LPS/TLR4 pathway. MS19 inhibits the phosphorylation of p65 NF-κB and release of HMGB1 induced by LPS (red solid line); MS19 does not inhibit the phosphorylation of TAK-1, ERK, and p38 MAPK (red cross); our previous study reported that MS19 inhibits the expression and nuclear translocation of IRF5 (red dotted line).

Article Snippet: After washing with PBS, cells were incubated with goat anti-rabbit IgG against HMGB1 conjugated with FITC (1:200; Beyotime) for 1 h at room temperature.

Techniques: Inhibition, Phospho-proteomics, Expressing, Translocation Assay

Journal: European journal of pharmacology

Article Title: HMGB1 promoted P-glycoprotein at the blood-brain barrier in MCAO rats via TLR4/NF-κB signaling pathway.

doi: 10.1016/j.ejphar.2020.173189

Figure Lengend Snippet: Fig. 2. Upregulation of HMGB1, TLR4, pIKBα and GFAP in astrocytes and brain microvessel endothelial cells in MCAO rats. (A, B and D) Compared with the sham group, the expression of HMGB1, TLR4, pIKBα and GFAP was potentiated in the cerebral cortex tissues of MCAO rats. The scale bars represent 60 and 30 μm in (A) and (B) respectively. (C and E) Consistently, the HMGB1 (red) in astrocytes in MCAO group translocated from nucleus (Blue color represents DAPI) to the cytoplasm (Green color represents GFAP). The expression of TLR4 (yellow) and pIKBα (yellow) in brain microvessel endothelial cells (Green color represents VIII factor) in MCAO group was potentiated respectively. The expression of GFAP (green) in astrocytes (Blue color represents DAPI) in MCAO group was also increased. The scale bars represent 10 μm. (D and E) Each value represents the mean ± S.D. (n = 10) and was measured as described in “Materials and Methods”. Note: *P < 0.05 vs. sham group.

Article Snippet: Brain slices were treated with antibodies against HMGB1 (rabbit polyclonal IgG antibody; 1:100), TLR4 (rabbit polyclonal IgG antibody; 1:100), pIKBα (rabbit polyclonal IgG antibody; 1:100) and glial fibrillary acidic protein (GFAP) (rabbit polyclonal IgG antibody; 1:100) at 4 °C and reacted with avidin-biotin-peroxidase complex and DAB (Boster Bio-engineering, Wuhan, China).

Techniques: Expressing

Journal: European journal of pharmacology

Article Title: HMGB1 promoted P-glycoprotein at the blood-brain barrier in MCAO rats via TLR4/NF-κB signaling pathway.

doi: 10.1016/j.ejphar.2020.173189

Figure Lengend Snippet: Fig. 6. OGD activated astrocytes and induced HMGB1 release into the extracellular space. (A and C) OGD potentiated GFAP expression in astrocytes. GFAP was labelled with HRP (yellow) and nucleus was stained with DAPI (blue). (B (a and b), C, D, E and F) OGD increased HMGB1 expression (red) in astrocytes and induced HMGB1 release from nucleus to the cytoplasm. GFAP was labelled with FITC (green) and nucleus was stained with DAPI (blue). (B (c and d)) Expression of HMGB1 was not observed in rBMECs in both sham and OGD groups. VIII factor was labelled with FITC (green) and nucleus was stained with DAPI (blue). (G) OGD increased HMGB1 content in the medium. (C, D (b), E (b), F (b) and G (b)) Each value represents the mean ± S.D. (n = 10) and was measured as described in “Materials and Methods”. Note: *P < 0.05 vs. sham group. Scale bars represent 20 μm.

Article Snippet: Brain slices were treated with antibodies against HMGB1 (rabbit polyclonal IgG antibody; 1:100), TLR4 (rabbit polyclonal IgG antibody; 1:100), pIKBα (rabbit polyclonal IgG antibody; 1:100) and glial fibrillary acidic protein (GFAP) (rabbit polyclonal IgG antibody; 1:100) at 4 °C and reacted with avidin-biotin-peroxidase complex and DAB (Boster Bio-engineering, Wuhan, China).

Techniques: Expressing, Staining

Journal: European journal of pharmacology

Article Title: HMGB1 promoted P-glycoprotein at the blood-brain barrier in MCAO rats via TLR4/NF-κB signaling pathway.

doi: 10.1016/j.ejphar.2020.173189

Figure Lengend Snippet: Fig. 8. HMGB1 contributed to upregulation of P-gp via TLR4/NF-kB pathway. (A) The relationship among HMGB1 and changes of TLR4, TIRAP, NF-kB and P-gp in rBMECs after OGD was investigated using related positive agents. (B) Changes of TLR4 (green) and TIRAP (red) after OGD and treatment with positive agents in rBMECs. (C) Each value represents the mean ± S.D. (n = 10) and was measured as described in “Materials and Methods”. Note: *P < 0.05 vs. sham group; #P < 0.05 vs. OGD group. The scale bars represent 10 μm.

Article Snippet: Brain slices were treated with antibodies against HMGB1 (rabbit polyclonal IgG antibody; 1:100), TLR4 (rabbit polyclonal IgG antibody; 1:100), pIKBα (rabbit polyclonal IgG antibody; 1:100) and glial fibrillary acidic protein (GFAP) (rabbit polyclonal IgG antibody; 1:100) at 4 °C and reacted with avidin-biotin-peroxidase complex and DAB (Boster Bio-engineering, Wuhan, China).

Techniques:

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: The Journal of Cell Biology

Article Title: Extracellular HMGB1, a signal of tissue damage, induces mesoangioblast migration and proliferation

doi: 10.1083/jcb.200304135

Figure Lengend Snippet: Effect of HMGB1 on embryonic mesoangioblast proliferation. (A) D16 cells were grown in RPMI medium containing no addition, HMGB1 at the indicated concentrations, or 20% FCS. HMGB1 induced cell proliferation at all concentrations tested, but the cell number reached a plateau after 48 h. Each point represents the mean ± SD ( n = 3). The experiment was repeated three times. (B) D16 cell division was analyzed by FACS ® . After 6 h in the presence of 30 ng/ml HMGB1 the DNA content increases, but returns to the normal diploid content after 24 h. Asterisk indicates statistical significance (P < 0.001). (C) 3T3 fibroblasts (treated as the D16 cells in A) do not divide in the presence of HMGB1.

Article Snippet: Serial muscle sections were fixed in 4% PFA, permeabilized, saturated, and processed for immunofluorescence with rabbit anti-HMGB1 antibody (1:500 dilution) followed by Alexa Fluor ® 488–conjugated goat anti–rabbit Ig (Molecular Probes, Inc.).

Techniques:

Journal: The Journal of Cell Biology

Article Title: Extracellular HMGB1, a signal of tissue damage, induces mesoangioblast migration and proliferation

doi: 10.1083/jcb.200304135

Figure Lengend Snippet: Continued HMGB1 stimulation sustains mesoangioblast proliferation. D16 cells were placed at time 0 in RPMI medium containing 30 ng/ml HMGB1; a similar amount of HMGB1 was also added at the times indicated with a triangle. Multiply stimulated D16 cells kept growing. Each point represents the average ± SD of two experiments performed in duplicate. Inset: Western blot of HMGB1 in the medium bathing D16 cells 48 h after the beginning of the experiment. HMGB1 was still present in the medium of restimulated cells, but not in the medium of cells stimulated once at time 0. This experiment was repeated two times with similar results.

Article Snippet: Serial muscle sections were fixed in 4% PFA, permeabilized, saturated, and processed for immunofluorescence with rabbit anti-HMGB1 antibody (1:500 dilution) followed by Alexa Fluor ® 488–conjugated goat anti–rabbit Ig (Molecular Probes, Inc.).

Techniques: Western Blot

Journal: The Journal of Cell Biology

Article Title: Extracellular HMGB1, a signal of tissue damage, induces mesoangioblast migration and proliferation

doi: 10.1083/jcb.200304135

Figure Lengend Snippet: HMGB1 has chemotactic activity on embryonic mesoangioblasts. (A) D16 cells were subjected to chemotaxis assays with 10, 50, or 100 ng/ml HMGB1. Data represent the average ± SD of four experiments performed in duplicate; the effect of increasing HMGB1 concentrations is highly significant (P < 0.001 in ANOVA analysis). Addition of anti-HMGB1 antibodies recognizing the peptide 166–181 significantly reduced the chemotactic response (P < 0.05 in comparison to the sample without antibody), whereas the addition of monoclonal anti-box A antibodies had no effect. (B) Chemotactic activity on D16 cells of various HMGB1 fragments (all at 10 ng/ml). ABbt has a chemotactic effect comparable with full-length HMGB1 (P < 0.05 of proteins vs. medium alone). In contrast, boxes A and B and the AB didomain have no significant chemotactic activity. Bars represent the average ± SD of three experiments performed in duplicate. Asterisks indicate statistical significance (P< 0.01). (C) Schematic representation of full-length HMGB1, boxes A and B, the didomain AB, and tailless HMGB1 (ABbt). (D) Western blot with anti-RAGE antibodies on total D16 cell extract. (E) D16 cells transfected with dnRAGE-expressing plasmid or pCDNA3 empty vector were assayed for chemotaxis in response to medium with or without HMGB1. Cells transfected with dnRAGE exhibited a significant decrease in migration in comparison to cells transfected with control plasmid. Asterisks indicate statistical significance (P < 0.05).

Article Snippet: Serial muscle sections were fixed in 4% PFA, permeabilized, saturated, and processed for immunofluorescence with rabbit anti-HMGB1 antibody (1:500 dilution) followed by Alexa Fluor ® 488–conjugated goat anti–rabbit Ig (Molecular Probes, Inc.).

Techniques: Activity Assay, Chemotaxis Assay, Western Blot, Transfection, Expressing, Plasmid Preparation, Migration

Journal: The Journal of Cell Biology

Article Title: Extracellular HMGB1, a signal of tissue damage, induces mesoangioblast migration and proliferation

doi: 10.1083/jcb.200304135

Figure Lengend Snippet: HMGB1 induces the transit of mesoangioblasts through an endothelial monolayer. D16 cells were placed in the upper compartment of Boyden apparatuses. The lower chambers contained RPMI alone (medium), RPMI plus 100 ng/ml HMGB1, or RPMI plus 10 ng/ml VEGF; chambers were separated by a confluent endothelial cell monolayer grown on polycarbonate filters. HMGB1 significantly stimulated D16 transmigration (P < 0.01). Bars represent the aver- age ± SD of three experiments performed in duplicate. Panels beside the bar graph show D16 cells stained with Giemsa after migration, toward medium alone or containing HMGB1. Asterisk indicates statistical significance (P < 0.05).

Article Snippet: Serial muscle sections were fixed in 4% PFA, permeabilized, saturated, and processed for immunofluorescence with rabbit anti-HMGB1 antibody (1:500 dilution) followed by Alexa Fluor ® 488–conjugated goat anti–rabbit Ig (Molecular Probes, Inc.).

Techniques: Transmigration Assay, Staining, Migration

Journal: The Journal of Cell Biology

Article Title: Extracellular HMGB1, a signal of tissue damage, induces mesoangioblast migration and proliferation

doi: 10.1083/jcb.200304135

Figure Lengend Snippet: HMGB1 attracts mesoangioblasts in vivo. D16 cells were first transduced with a lentiviral vector encoding nuclear LacZ, and were then injected through the femoral artery of mice where heparin-Sepharose beads (either unloaded or loaded with HMGB1) had been injected in the tibialis anterior muscle. Mice were killed after 24 h. (A) Tibialis anterior muscles injected with HMGB1-loaded and control beads. (B–D) Cryosections of muscles treated with control heparin-Sepharose beads (control) or HMGB1-coated heparin-Sepharose beads (HMGB1). Arrows indicate the beads. Sections were stained with X-gal, and mesoangioblasts (arrowheads) appear blue. Mesoangioblasts were found in large clusters (C) or as isolated cells (D) only in muscles injected with HMGB1-coated beads. (E) Number of migrating D16 cells in tibialis anterior muscles of wild-type mice treated with HMGB1-loaded beads (gray bar; n = 2) or control beads coinjected with 1 μg LPS (black bar; n = 3). White bar represents the number of D16 cells found in tibialis anterior muscles of α-SG −/− dystrophic mice ( n = 2).

Article Snippet: Serial muscle sections were fixed in 4% PFA, permeabilized, saturated, and processed for immunofluorescence with rabbit anti-HMGB1 antibody (1:500 dilution) followed by Alexa Fluor ® 488–conjugated goat anti–rabbit Ig (Molecular Probes, Inc.).

Techniques: In Vivo, Transduction, Plasmid Preparation, Injection, Staining, Isolation

Journal: The Journal of Cell Biology

Article Title: Extracellular HMGB1, a signal of tissue damage, induces mesoangioblast migration and proliferation

doi: 10.1083/jcb.200304135

Figure Lengend Snippet: Effect of HMGB1 on adult mesoangioblasts. (A) Mesoangioblasts of the G1 clone, obtained from mouse bone marrow, were grown in RPMI medium containing 1, 10, or 30 ng/ml HMGB1. For comparison, G1 cells were also grown in RPMI medium alone or in RPMI plus 20% FCS. (B) Migration of G1 cells toward the lower chamber of Boyden apparatuses containing RPMI (medium) or RPMI plus 10 ng/ml HMGB1 (HMGB1). In the migration experiment, the chambers were separated by a filter; in the transmigration experiment, the chambers were separated by a filter overgrown with a monolayer of endothelial cells. Each bar represents the average ± SD of three experiments, and the arrows indicate statistical significance (P < 0.05). (C) G1 cells were labeled with DiI and then injected through the femoral artery of mice where heparin-Sepharose beads (either loaded with HMGB1 or unloaded) had been implanted in the tibialis anterior muscle (arrows). Top, phase contrast; bottom, fluorescence. G1 cells (red fluorescence) migrate in the vicinity of HMGB1-loaded cells; no G1 cells are detected near control beads.

Article Snippet: Serial muscle sections were fixed in 4% PFA, permeabilized, saturated, and processed for immunofluorescence with rabbit anti-HMGB1 antibody (1:500 dilution) followed by Alexa Fluor ® 488–conjugated goat anti–rabbit Ig (Molecular Probes, Inc.).

Techniques: Migration, Transmigration Assay, Labeling, Injection, Fluorescence

Journal: The Journal of Cell Biology

Article Title: Extracellular HMGB1, a signal of tissue damage, induces mesoangioblast migration and proliferation

doi: 10.1083/jcb.200304135

Figure Lengend Snippet: HMGB1 expression and mesoangioblast migration in α -SG − / − dystrophic muscles. (A) Western blot analysis of HMGB1 expression levels in tibialis anterior muscles from α-SG −/− dystrophic and wild-type mice. (B) Hematoxylin and eosin staining (H&E) of tibialis anterior muscle sections from α-SG −/− dystrophic and wild-type mice. Different sections from the same muscles were processed for immunofluorescence (right). A considerable number of foci containing inflammatory cells were evident in α-SG −/− dystrophic muscles, and these contained HMGB1 in the cytoplasm in addition to the nucleus (anti-HMGB1, green; DAPI, red pseudocolor). (C) Fraction of dnRAGE expressing D16 cells before and after injection and homing to tibialis anterior muscles of α-SG −/− dystrophic mice. About 500 cells were counted before and after injection in two mice. The difference before and after migration is not statistically different. (D) Number of migrating D16 cells in tibialis anterior muscles of α-SG −/− dystrophic mice injected with control beads ( n = 2) or HMGB1-loaded beads ( n = 2). Cells expressing dnRAGE and GFP are indicated in green; the nonexpressing cells are indicated in gray. The difference in total number of cells, and in cells not expressing dnRAGE, is significant (P < 0.05). The difference in cells expressing dnRAGE is not statistically significant.

Article Snippet: Serial muscle sections were fixed in 4% PFA, permeabilized, saturated, and processed for immunofluorescence with rabbit anti-HMGB1 antibody (1:500 dilution) followed by Alexa Fluor ® 488–conjugated goat anti–rabbit Ig (Molecular Probes, Inc.).

Techniques: Expressing, Migration, Western Blot, Staining, Immunofluorescence, Injection