Journal: The Journal of biological chemistry

Article Title: DNA-mediated proteolysis by neutrophil elastase enhances binding activities of the HMGB1 protein.

doi: 10.1016/j.jbc.2022.102577

Figure Lengend Snippet: Figure 6. Potential roles of the DNA-mediated proteolytic processing of HMGB1 by neutrophil elastase in NETs. Due to the enhanced binding activities of the processed HMGB1 protein, this processing may promote (1) TLR4 signaling, (2) binding to biofilm DNA, and (3) DNA sensing by cGAS. Due to the loss of residues 177–215, the processing of HMGB1 may diminish (4) RAGE signaling and (5) nuclear localization. NET, neutrophil extracellular trap.

Article Snippet: Lyophilized TLR4 MD-2 complex was purchased from R&D Systems (catalog no.: #3146-TM-050).

Techniques: Binding Assay

Journal: Kidney international

Article Title: Poly(ADP-ribose) polymerase 1 activation is required for cisplatin nephrotoxicity.

doi: 10.1038/ki.2012.64

Figure Lengend Snippet: Figure 5 | Poly(ADP-ribose) polymerase 1 (Parp1) deficiency inhibits inflammation induced by cisplatin injection in kidneys. The kidneys in Parp1-knockout (KO) and -wild-type (WT) male mice were harvested 0, 1, 2, 3, or 5 days after cisplatin (20 mg/kg body weight) or saline (control) injection. (a) Polymorphonuclear neutrophil (PMN)–positive cells were counted in 10 fields ( 200 magnification) per kidney (n ¼ 4 in each group). (b) The expression of intercellular adhesion molecule-1 (ICAM-1), tumor necrosis factor-a (TNF-a), and Toll-like receptor 4 (TLR4) in kidneys was examined by western blot analysis. Anti-b-actin antibody was used as a loading control. (c–e) The intensities of protein bands (ICAM-1, 110 kDa; TNF-a, 26 kDa; TLR4, 90 kDa; all approximate) were quantified using the Lab Works analysis software (n ¼ 6 in each group). *Po0.05 vs. 0 days, #Po0.05 vs. WT.

Article Snippet: Western blot analysis was confirmed, as described previously,62–64 using antibodies against PARP1, cleaved caspase-3, phospho-RelA, phospho-p38, phospho-38, phospho-JNK, JNK, phospho-MKK3/6, phospho-MKK4 (Cell Signaling, Beverly, MA), ICAM-1, RelA, IkBa (Santa Cruz, Santa Cruz, CA), PAR (BD Pharmingen, San Jose, CA), b-actin (Sigma, St. Louis, MO), TNF-a (Abcam, Cambridge, MA), or TLR4 (Imgenex, San Diego, CA).

Techniques: Injection, Knock-Out, Saline, Control, Expressing, Western Blot, Software

Journal: Kidney international

Article Title: Poly(ADP-ribose) polymerase 1 activation is required for cisplatin nephrotoxicity.

doi: 10.1038/ki.2012.64

Figure Lengend Snippet: Figure 8 | Poly(ADP-ribose) polymerase 1 (Parp1) deficiency blocks TLR4/MAPK/NF-jB/TNF-a signaling pathway during necrotic cell death induced by cisplatin treatment in primary culture of proximal tubule epithelial cells. The proximal tubule epithelial cells were isolated from Parp1-knockout (KO) and -wild-type (WT) mouse kidneys. After 18 h of starvation, the cells were treated with 400 mmol/l cisplatin for 4 h. Dimethyl sulfoxide (DMSO) was used as vehicle. To downregulate Toll-like receptor 4 (TLR4) expression, 500 pmol of small interference RNA (siRNA) was transfected on a 100-mm culture dish for 24 h before cisplatin or vehicle treatment. The same concentration of scramble siRNA was used as control. Pharmacological inhibitors (100 mmol/l of SB203580 against p38 activation, 10 mmol/l of SP600125 against c-Jun N-terminal kinase (JNK) activation, 100 mmol/l of ammonium pyrrolidinedithiocarbamate against nuclear factor-kB (NF-kB) activation, or 100 mmol/l of thalidomide against tumor necrosis factor-a (TNF-a) synthesis) or the same volume of DMSO (control) were added 2 h before cisplatin or vehicle treatment. Results are representative of experiments repeated three times.

Article Snippet: Western blot analysis was confirmed, as described previously,62–64 using antibodies against PARP1, cleaved caspase-3, phospho-RelA, phospho-p38, phospho-38, phospho-JNK, JNK, phospho-MKK3/6, phospho-MKK4 (Cell Signaling, Beverly, MA), ICAM-1, RelA, IkBa (Santa Cruz, Santa Cruz, CA), PAR (BD Pharmingen, San Jose, CA), b-actin (Sigma, St. Louis, MO), TNF-a (Abcam, Cambridge, MA), or TLR4 (Imgenex, San Diego, CA).

Techniques: Isolation, Knock-Out, Expressing, Transfection, Concentration Assay, Control, Activation Assay

Journal: Kidney international

Article Title: Poly(ADP-ribose) polymerase 1 activation is required for cisplatin nephrotoxicity.

doi: 10.1038/ki.2012.64

Figure Lengend Snippet: Figure 11 | Poly(ADP-ribose) polymerase 1 (PARP1) inhibitor reduces inflammation induced by cisplatin injection in kidneys. The kidneys in 129S1/SvImJ male mice were harvested 0, 3, or 5 days after cisplatin injection. The mice were administered either PJ34 (10 mg/kg body weight) or saline (control, Con) twice daily intraperitoneally from 24 h before cisplatin injection up to the time that they were killed. (a) Polymorphonuclear neutrophil (PMN)-positive cells were counted in 10 fields ( 200 magnification) per kidney (n ¼ 4 in each group). (b) The expression of Poly(ADP-ribose) (PAR), intercellular adhesion molecule-1 (ICAM-1), tumor necrosis factor-a (TNF-a), and Toll-like receptor 4 (TLR4) in kidneys was examined by western blot analysis. Anti-b-actin antibody was used as a loading control. (c–f) The intensities of protein bands (PAR, 116 kDa; ICAM-1, 110 kDa; TNF-a, 26 kDa; TLR4, 90 kDa; all approximate) were quantified using the Lab Works analysis software (n ¼ 4 in each group). *Po0.05 vs. 0 days; #Po0.05 vs. control.

Article Snippet: Western blot analysis was confirmed, as described previously,62–64 using antibodies against PARP1, cleaved caspase-3, phospho-RelA, phospho-p38, phospho-38, phospho-JNK, JNK, phospho-MKK3/6, phospho-MKK4 (Cell Signaling, Beverly, MA), ICAM-1, RelA, IkBa (Santa Cruz, Santa Cruz, CA), PAR (BD Pharmingen, San Jose, CA), b-actin (Sigma, St. Louis, MO), TNF-a (Abcam, Cambridge, MA), or TLR4 (Imgenex, San Diego, CA).

Techniques: Injection, Saline, Control, Expressing, Western Blot, Software

Journal: The American Journal of Pathology

Article Title: Enhanced Susceptibility to Endotoxic Shock and Impaired STAT3 Signaling in CD31-Deficient Mice

doi: 10.1016/s0002-9440(10)62243-2

Figure Lengend Snippet: Figure 11. TLR4 expression of WT and CD31-deficient splenocytes and endothelial cells remains constant and does not change following LPS treat- ment. Representative FACS analyses of WT (A) and KO (B) splenocytes in the absence and presence of LPS; KO (C) and PECAM-1 reconstituted (D) lung endothelial cells and KO (E) and WT (F) brain endothelial cells illus- trating essentially no differences in TLR4 expression. This was confirmed by Western blot analyses (data not shown).

Article Snippet: Splenocytes were prepared as described, fixed in 2% PFA, then stained with 0.5 g of polyclonal anti-mouse TLR4 (Imgenex) in Hanks’ buffered salt solution plus 1% BSA for 1 hour on ice, then, following two washes, in donkey anti-rabbit IgG F(ab )2 PE conjugate (Jackson Immunoresearch, West Grove, PA).

Techniques: Expressing, Western Blot

Journal: Biochemical and biophysical research communications

Article Title: Platelet-derived high-mobility group box 1 promotes recruitment and suppresses apoptosis of monocytes

doi: 10.1016/j.bbrc.2016.07.078

Figure Lengend Snippet: HMGB1 inhibits monocyte apoptosis induced by ABT-737 (A,C) or staurosporine (B,D), which is reversed when monocytes are pretreated with a blocking TLR4 antibody, as evaluated by Annexin V (A,B) and TMRE (C,D) stainings. (E) U0126, a specific MEK/ERK inhibitor, inhibits the effect of rHMGB1 on TMRE fluorescence in monocytes. (F) rHMGB1 (100 ng/ml) induces phosphorylation of ERK in monocytes, which does not occur when monocytes are pretreated with a blocking TLR4 antibody. Data are presented as mean ± SD for N≥4 and at least three separate experiments in all studies. * p<0.05, # p<0.05, ** p<0.01 (Student’s t test).

Article Snippet: When indicated, HMGB1 receptors were blocked on monocytes with anti-human RAGE polyclonal antibody (20 μg/ml, goat IgG), anti-human TLR2 monoclonal antibody (2 μg/ml, mouse IgG2b) or anti-human TLR4 polyclonal antibody (10 μg/ml, goat IgG) (R&D Systems, Wiesbaden, Germany).

Techniques: Blocking Assay, Fluorescence, Phospho-proteomics

Journal: Journal of Cellular and Molecular Medicine

Article Title: Up‐regulated TLR 4 in cardiomyocytes exacerbates heart failure after long‐term myocardial infarction

doi: 10.1111/jcmm.12659

Figure Lengend Snippet: Increased toll‐like receptor 4 ( TLR 4) expression in the myocardium of chronic heart failure ( CHF ) rats. ( A ) TLR 4 mRNA levels in infarct and remote myocardium of sham and CHF rats (n = 6/group). ( B ) Representative Western blot images and ( C ) quantification of TLR 4 proteins in infarct and remote myocardium of sham and CHF rats (n = 4/group). ( D ) Representative immunohistochemistry images of heart sections stained with TLR 4 (green) and CD 45 (red). The yellow box indicates the enlarged area shown on the right (data are means ± SD , * P < 0.05, ** P < 0.01 versus respective sham).

Article Snippet: The membrane was then blocked with 5% non‐fat dried milk, and probed with the primary antibody against TLR4 (Cat. NB100‐56566; Novus Biologicals, Littleton, CO, USA) followed by the peroxidase‐conjugated secondary antibody, at the concentration of 1:500 and 1:1000 respectively.

Techniques: Expressing, Western Blot, Immunohistochemistry, Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: Up‐regulated TLR 4 in cardiomyocytes exacerbates heart failure after long‐term myocardial infarction

doi: 10.1111/jcmm.12659

Figure Lengend Snippet: Increased toll‐like receptor 4 ( TLR 4) expression in the surviving cardiomyocytes of chronic heart failure ( CHF ) rats. ( A ) Representative immunofluorescent images of TLR 4 in cardiomyocytes isolated from sham and CHF rats. ( B ) TLR 4 mRNA levels in cardiomyocytes isolated from sham and CHF rats. ( C ) Representative Western blot images and ( D ) quantification of TLR 4 proteins in cardiomyocytes isolated from sham and CHF rats (data are means ± SD , n = 6/group, ** P < 0.01 versus sham).

Article Snippet: The membrane was then blocked with 5% non‐fat dried milk, and probed with the primary antibody against TLR4 (Cat. NB100‐56566; Novus Biologicals, Littleton, CO, USA) followed by the peroxidase‐conjugated secondary antibody, at the concentration of 1:500 and 1:1000 respectively.

Techniques: Expressing, Isolation, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Up‐regulated TLR 4 in cardiomyocytes exacerbates heart failure after long‐term myocardial infarction

doi: 10.1111/jcmm.12659

Figure Lengend Snippet: Toll‐like receptor 4 ( TLR 4)‐sh RNA lentivirus reduced myocardial inflammation and improved heart function after myocardial infarction ( MI ). The rats received intra‐myocardial injection of normal saline ( NS ), control‐sh RNA lentivirus or TLR 4‐sh RNA lentivirus (1 × 10 9 TU /ml, 100 μl/heart) just after left anterior descending coronary artery ( LAD ) ligation or sham operation. All examinations were performed after 4 weeks of MI . ( A ) Expression of green fluorescent protein ( GFP ; green), the marker gene carried by TLR 4‐sh RNA lentivirus, in the myocardium. The nuclei were counter‐stained with Hoechst 33258 (blue). ( B ) Representative Western blot images and quantification of TLR 4 proteins in sham and chronic heart failure ( CHF ) myocardium. ( C ) tumour necrosis factor ( TNF )‐α and interleukin ( IL )‐6 protein contents in infarct and remote myocardium. ( D ) Representative images of Masson's trichrome staining (upper panel) and quantification (lower panel) of post‐infarct failing hearts, showing that TLR 4‐sh RNA lentivirus reduced cardiac fibrosis. Cross‐sections were cut at the midhorizontal plane of the fixed paraffin‐embedded heart, and stained with Masson's trichrome reagents. ( E ) Infarct size of post‐infarct failing hearts. ( F ) Fractional shortening (%) of the left ventricle (data are means ± SD , n = 4/group, a P < 0.05, A P < 0.01 versus respective sham‐ NS ; B P < 0.01 versus respective CHF ‐ NS ).

Article Snippet: The membrane was then blocked with 5% non‐fat dried milk, and probed with the primary antibody against TLR4 (Cat. NB100‐56566; Novus Biologicals, Littleton, CO, USA) followed by the peroxidase‐conjugated secondary antibody, at the concentration of 1:500 and 1:1000 respectively.

Techniques: Injection, Saline, Control, Ligation, Expressing, Marker, Staining, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Up‐regulated TLR 4 in cardiomyocytes exacerbates heart failure after long‐term myocardial infarction

doi: 10.1111/jcmm.12659

Figure Lengend Snippet: Enhanced binding activity of toll‐like receptor 4 ( TLR 4) in chronic heart failure ( CHF ) cardiomyocytes to lipopolysaccharide ( LPS ) and heat shock protein 60 ( HSP 60). Isolated cardiomyocytes were cultured in a CO 2 incubator at 37°C for 24 hrs, then the binding assay was performed at 4°C for 30 min. To block TLR 4, cultured cardiomyocytes were incubated with TLR 4 neutralizing antibody (anti‐ TLR 4, 5 μg/ml) at 37°C for 15 min., and subsequently incubated with FITC ‐ LPS or OG ‐ HSP 60 at 4°C for 30 min. ( A ) Representative fluorescent images of isolated cardiomyocytes after the incubation with FITC ‐ LPS (green) or OG ‐ HSP 60 (green). ( B ) Binding curves of FITC ‐ LPS to cardiomyocytes. ( C ) Binding curves of OG ‐ HSP 60 to cardiomyocytes.

Article Snippet: The membrane was then blocked with 5% non‐fat dried milk, and probed with the primary antibody against TLR4 (Cat. NB100‐56566; Novus Biologicals, Littleton, CO, USA) followed by the peroxidase‐conjugated secondary antibody, at the concentration of 1:500 and 1:1000 respectively.

Techniques: Binding Assay, Activity Assay, Isolation, Cell Culture, Blocking Assay, Incubation

Journal: Journal of Cellular and Molecular Medicine

Article Title: Up‐regulated TLR 4 in cardiomyocytes exacerbates heart failure after long‐term myocardial infarction

doi: 10.1111/jcmm.12659

Figure Lengend Snippet: Increased cytokine production mediated by toll‐like receptor 4 ( TLR 4) in chronic heart failure ( CHF ) cardiomyocytes. Cultured cardiomocytes from sham and CHF rats were treated with lipopolysaccharide ( LPS ; 1 μg/ml) or heat shock protein 60 ( HSP 60; 1 μg/ml) for 6 hrs. TLR 4 neutralizing antibody (anti‐ TLR 4, 5 μg/ml) was added 15 min before LPS or HSP 60 treatment. ( A ) Tumour necrosis factor ( TNF )‐α and interleukin ( IL )‐6 mRNA levels (n = 6/group). ( B ) The amount of TNF ‐α and IL ‐6 released into culture supernatant (n = 6/group). ( C ) Representative Western blot images and quantification of p65 in the nuclei of cardiomyocytes from three independent experiments (data are means ± SD , a P < 0.05, A P < 0.01 versus respective sham; b P < 0.05, B P < 0.01 versus sham‐blank; c P < 0.05, C P < 0.01 versus CHF ‐blank; d P < 0.05, D P < 0.01 versus respective LPS ; e P < 0.05, E P < 0.01 versus respective HSP60).

Article Snippet: The membrane was then blocked with 5% non‐fat dried milk, and probed with the primary antibody against TLR4 (Cat. NB100‐56566; Novus Biologicals, Littleton, CO, USA) followed by the peroxidase‐conjugated secondary antibody, at the concentration of 1:500 and 1:1000 respectively.

Techniques: Cell Culture, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Up‐regulated TLR 4 in cardiomyocytes exacerbates heart failure after long‐term myocardial infarction

doi: 10.1111/jcmm.12659

Figure Lengend Snippet: Primers for real‐time PCR

Article Snippet: The membrane was then blocked with 5% non‐fat dried milk, and probed with the primary antibody against TLR4 (Cat. NB100‐56566; Novus Biologicals, Littleton, CO, USA) followed by the peroxidase‐conjugated secondary antibody, at the concentration of 1:500 and 1:1000 respectively.

Techniques:

Journal: Nature communications

Article Title: Mapping tenascin-C interaction with toll-like receptor 4 reveals a new subset of endogenous inflammatory triggers.

doi: 10.1038/s41467-017-01718-7

Figure Lengend Snippet: Fig. 1 The FBG domains of tenascin-C, -R, and -W can induce NF-kB activation and cytokine synthesis, and bind to TLR4. a Tenascin-C, -R, -W, and -X each contain an assembly domain, a variable number of epidermal growth factor (EGF)-like repeats, a variable number of fibronectin type III-like repeats (these can be constitutively expressed (white rectangles) or alternatively spliced (gray rectangles) and a C-terminal fibrinogen-like globe (FBG) domain. The FBG domains exhibit a similar molecular weight, comprising between 229 and 240 amino acids each (FBG-C: 26.1 kDa, amino acids 1974–2201, FBG-R: 27.0 kDa, amino acids 1128–1359, FBG-W: 27.5 kDa, amino acids 1060–1300, FBG-X: 26.1 kDa, amino acids 4013–4243); protein accession numbers: tenascin-C (P24821), tenascin-R (Q92752), tenascin-W (Q9UQP3), tenascin-X (P22105). b THP1 NF-kB cells were stimulated with different concentrations of FBG-C, -R, -W, and -X, or were left unstimulated (−) for 24 h and NF-kB activation measured using QUANTI-Blue. Data are shown as mean ± SEM from three independent experiments. One-way ANOVA vs. non-stimulated, **p < 0.01, ***p < 0.001. c–e Primary human macrophages were stimulated with different concentrations of FBG-C,-R, -W, and -X, or were left unstimulated (−) for 24 h, and TNF (c), IL-6 (d), and IL-8 (e) levels measured by ELISA. Data are shown as mean ± SEM from three independent donors. One-way ANOVA vs. non-stimulated, *p < 0.05, **p < 0.01, ***p < 0.001. f 96-well plates were coated with 1 µg ml−1 of FBG-C, -R, -W, or -X, or PBS, and incubated with increasing doses of TLR4. Curves were fitted in GraphPad Prism using one-binding site hyperbola equation. Data in the graph are shown as mean ± SEM from four independent experiments

Article Snippet: Recombinant human TLR4 was purchased from R&D systems.

Techniques: Activation Assay, Molecular Weight, Enzyme-linked Immunosorbent Assay, Incubation, Binding Assay

Journal: Nature communications

Article Title: Mapping tenascin-C interaction with toll-like receptor 4 reveals a new subset of endogenous inflammatory triggers.

doi: 10.1038/s41467-017-01718-7

Figure Lengend Snippet: Fig. 2 Peptide mapping reveals specific regions in FBG-C involved in TLR4 activation and binding. a Nine peptides of ~30 amino acids long from FBG-C were synthesized; overlapping amino acid sequences are shown in bold. b THP1 NF-kB cells were stimulated with LPS (0.5 ng ml−1), FBG-C (0.5 µM), or 20, 50, or 100 µM of peptides 1–9 for 24 h and NF-kB activation was measured using QUANTI-Blue™. Data shown as mean ± SEM, n = 4 independent experiments. One-way ANOVA vs. unstimulated cells. **p < 0.01, ***p < 0.001. c Increasing doses of TLR4 were pre-incubated with 200 µM of peptides before adding them to 96-well plates coated with 1 µg ml−1 of FBG-C. Curves were fitted in GraphPad Prism using one-binding site hyperbola equation. Data are shown as mean ± SEM, n = 3. d THP1 NF-kB cells were left unstimulated (−) or pre-incubated with 100 µM peptides prior to stimulation with 0.5 µM of FBG-C for 24 h. NF-kB activation was measured using QUANTI-Blue™. Data shown as mean ± SEM, n = 3 independent experiments. Paired t-test vs. FBG- C only, *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: Recombinant human TLR4 was purchased from R&D systems.

Techniques: Activation Assay, Binding Assay, Synthesized, Incubation

Journal: Nature communications

Article Title: Mapping tenascin-C interaction with toll-like receptor 4 reveals a new subset of endogenous inflammatory triggers.

doi: 10.1038/s41467-017-01718-7

Figure Lengend Snippet: Fig. 4 Pinpointing amino acids in loops 5, 7, and 10 of FBG-C that mediate TLR4 binding and activation. Upper panel: Sequences of wild-type FBG-C and mutants 1–7, highlighting wild-type amino acids in blue and mutations in red (loop 5 variants are shown in a, loop 10 in b, and loop 7 in c). Second panel: THP1 NF-kB cells were left unstimulated (−) or stimulated for 24 h with LPS (1 ng ml−1), increasing doses (μM) of FBG-C or FBG-C mutants 1–7. NF-kB activation was measured using QUANTI-Blue™. Data shown as mean ± SEM. n = 4 independent experiments. Paired t-test vs. FBG-C, *p < 0.05, **p < 0.01, ***p < 0.001. Third panel: Primary human macrophages were left unstimulated (−) or stimulated for 24 h with LPS (1 ng ml−1), increasing doses (μM) of FBG-C or FBG-C mutants 1–7. Cytokines synthesis was measured by ELISA. Data shown as mean ± SEM. n = 4 independent donors. Paired t-test vs. FBG-C, *p < 0.05, **p < 0.01, ***p < 0.001. Bottom panel: 96-well plates were coated with 1 µg ml−1 of FBG-C or FBG-C mutants 1–7, and TLR4 was added in a dose-dependent manner. Curves were fitted in GraphPad Prism using one-binding site hyperbola equation. Data shown as mean ± SEM; n = 4

Article Snippet: Recombinant human TLR4 was purchased from R&D systems.

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

Journal: Nature communications

Article Title: Mapping tenascin-C interaction with toll-like receptor 4 reveals a new subset of endogenous inflammatory triggers.

doi: 10.1038/s41467-017-01718-7

Figure Lengend Snippet: Fig. 5 Mutations in FBG-X confer TLR4-activating ability. a FBG-X chimeric proteins were designed to introduce the amino acids found in FBG-C to activate and bind to TLR4 (red) into the FBG-X sequence (blue). b ThP1 NF-kB cells were left unstimulated (−) or stimulated for 24 h with 0.5 ng ml−1 of LPS or increasing doses (μM) of FBG-C, FBG-X, FBG-X mutant 1, 2, 3, and 4. NF-kB activation was measured using QUANTI-Blue™. Data shown as mean ± SEM. n = 3 independent experiments. One-way ANOVA vs. FBG-C. c Primary human macrophages were left unstimulated (−) or stimulated for 24 h with 1 ng ml−1 of LPS or increasing doses (μM) of FBG-C, FBG-X, FBG-X mutant 1, 2, 3, and 4. Cytokine synthesis was measured by ELISA. Data shown as mean + SEM. n = 3 independent donors. One-way ANOVA vs. FBG-C. d 96-well plates were coated with 1 µg ml−1 of FBG-C, FBG-X, FBG-X mutant 1, 2, 3, and 4, and TLR4 was added in a dose-dependent manner. Data shown as mean ± SEM. n = 4 independent experiments

Article Snippet: Recombinant human TLR4 was purchased from R&D systems.

Techniques: Introduce, Sequencing, Mutagenesis, Activation Assay, Enzyme-linked Immunosorbent Assay

Journal: Nature communications

Article Title: Mapping tenascin-C interaction with toll-like receptor 4 reveals a new subset of endogenous inflammatory triggers.

doi: 10.1038/s41467-017-01718-7

Figure Lengend Snippet: Fig. 6 A conserved cationic ridge in fibrinogen-related proteins (FRePs). a Simplified domain organization of human FRePs: each protein contains distinct N-terminal sequences but all possess a C-terminal FBG domain, including the four tenascin family members (shown in Fig. 1a), α, β, and γ chains of fibrinogen, the three angiopoietins, seven of the angiopoietin-like proteins (Angio-LPs), the three ficolins, fibroleukin, FIBCD-1, FGL1, and MFAP4. b The cationic loop 5 ridge present in FBG-C, -R, and -W, but absent in FBG-X, is conserved in a subset of FRePs, which possess a comparable structural epitope made up of residues from loops 5, 6, and 7. Homology models of the FBG domains of the three FRePs selected for further analysis are shown together with that of tenascin-C (FBG-C); these include two predicted TLR4 agonists; the fibrinogen γ chain (FIB-G) and ficolin-1 (FIC-1), and one FBG domain predicted to be incapable of activating TLR4; angiopoietin-like protein 4 (ALP-4). The region created by residues from loops 5, 6, and 7 on the surface of each FBG domain is shown in pale orange, within which positively charged residues are colored red

Article Snippet: Recombinant human TLR4 was purchased from R&D systems.

Techniques:

Journal: Nature communications

Article Title: Mapping tenascin-C interaction with toll-like receptor 4 reveals a new subset of endogenous inflammatory triggers.

doi: 10.1038/s41467-017-01718-7

Figure Lengend Snippet: Fig. 7 The FBG domains of FIB-G and FIC-1 exhibit pro-inflammatory effects in vitro and in vivo. a–c Primary human macrophages were stimulated with different concentrations of FBG-C, FIB-G, FIC-1, and ALP-4, or were left unstimulated (−) for 24 h. Cytokine levels were measured by ELISA. Data shown as mean ± SEM from at least three independent donors. One-way ANOVA vs. non-stimulated, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. d Primary human macrophages were pre-incubated for 6 h with 3 µM TAK 242 prior to stimulation with FBG-C, FIB-G, FIC-1, and ALP-4 (1 µM), or no stimulation (−) for 24 h. Cytokine synthesis was measured by ELISA. Data shown as mean ± SEM from at least three independent donors. Paired t-test vs. non-treated, *p < 0.05, **p < 0.01, ***p < 0.001. e 96-well plates were coated with 1 µg ml−1 of FBG-C, FBG-X, FIB-G, FIC-1, and ALP-4, or PBS, and incubated with increasing doses of TLR4. Curves were fitted in GraphPad Prism using one-binding site hyperbola equation. Data are shown as mean ± SEM from three independent experiments. f, g Synovial inflammation was assessed 3 days post injection of each protein (1 µg) or PBS alone into the knees of DBA-1 mice. The histological score was calculated as the mean of seven sections from each knee joint per mouse. n = 5 mice per group except for FIC-1 (n = 4) (f). Mann–Whitney non-parametric test vs. PBS, *p < 0.05, **p < 0.01. Images show representative sections stained by haematoxylin and eosin (left panels) or safranin-O (right panels) (g). Mice injected with FBG-C, FIB-G, and FIC-1 exhibit cell infiltration into a thickened synovial lining layer, cellular invasion into the subchondral bone (arrows indicate bone erosion) and loss of articular cartilage proteoglycan (cp), pathological features not observed in mice injected with FBG-C mut or ALP-4.Scale bar left panels: 100 μM, right panels: 50 μM

Article Snippet: Recombinant human TLR4 was purchased from R&D systems.

Techniques: In Vitro, In Vivo, Enzyme-linked Immunosorbent Assay, Incubation, Binding Assay, Injection, MANN-WHITNEY, Staining

Journal: Nature communications

Article Title: Mapping tenascin-C interaction with toll-like receptor 4 reveals a new subset of endogenous inflammatory triggers.

doi: 10.1038/s41467-017-01718-7

Figure Lengend Snippet: Fig. 8 A common danger domain revealed. Three distinct sites within the FBG domain of tenascin-C contribute to TLR4 activation (center panel); a cationic ridge made up of residues from loops 5–7 (pale orange with positive residues highlighted red), underneath which sits a triad of hydrophobic/polar residues from loop 7 (green, purple, and blue), plus a C-terminal cationic tail in loop 10 (positive residues highlighted red). The cationic ridge is the dominant inflammatory epitope; its deletion renders inflammatory stimuli inert and its ectopic expression can convert immunologically inactive proteins into TLR4 agonists. In addition to tenascin-C, in other proteins that contain FBG domains, possession of this inflammatory epitope also confers TLR4-activating capabilities, irrespective of protein family (*denotes validated domains). Together, these data reveal a common mechanism by which distinct inflammatory triggers, spanning a wide range of tissue locations, induced in response to a spectrum of different threats, can activate TLR4 to raise an immune response

Article Snippet: Recombinant human TLR4 was purchased from R&D systems.

Techniques: Activation Assay, Expressing

Journal: Research

Article Title: Paeoniflorin Ameliorates Metabolic Dysfunction-Associated Steatotic Liver Disease by SYK/SH3BP2 Signaling Pathway

doi: 10.34133/research.1100

Figure Lengend Snippet: PF ameliorated HFFD-induced hepatic fibrosis progression in MASLD mice. (A and B) Representative images and quantification of hepatic Sirius scarlet staining. ( n = 3, scale bar = 50 μm). (C to E) Serum fibrosis factor HA, LN, and Col-IV levels. (F) Liver mRNA levels of α-SMA, Timp1, Tgf-β1, Col1a1, Fn1 , Pdgfrβ , and PAI-1 . (G and H) Western blot of α-SMA, TIMP1, and TGF-β1 protein expression and their quantification. Data were presented as the mean ± SEM ( n = 6). # P < 0.05, ## P < 0.01, ### P < 0.001 versus the ND group; * P < 0.05, ** P < 0.01, *** P < 0.001 versus the HFFD group.

Article Snippet: The antibodies used are as follows: SCD1 (2794T, CST, 1:1,000), PPAR-γ (66936-1-Ig, Proteintech, 1:2,000), IL-6 (12242T, CST, 1:1,000), TLR4 (66350-1-Ig, Proteintech, 1:3,000), TIMP1 (33502-1, SAB, 1:3,000), α-SMA (67735, Proteintech , 1:20,000), Tgf-β1 (26155, Proteintech, 1:1,000), α-tubulin (66031, Proteintech, 1:20,000), GAPDH (60004-1-IG, Proteintech, 1:6,000), Phospho-Syk (2710T, CST, 1:1,000), Syk (2712T, CST, 1:1,000), SH3BP2 (PA5-87739, Invitrogen, 1:1,000), Goat anti-Mouse IgG Secondary Antibody HRP (L3032, SAB, 1:5,000), and Goat anti-Rabbit IgG Secondary Antibody HRP (L3012, SAB, 1:5,000).

Techniques: Staining, Western Blot, Expressing

Journal: Research

Article Title: Paeoniflorin Ameliorates Metabolic Dysfunction-Associated Steatotic Liver Disease by SYK/SH3BP2 Signaling Pathway

doi: 10.34133/research.1100

Figure Lengend Snippet: PF alleviated MASLD fibrosis progression in vitro by inhibiting the SYK/SH3BP2 pathway. (A) LX2 cell survival ( n = 3). (B to G) Western blot of P-SYK, SYK, SH3BP2, α-SMA, TIMP1, and Tgf-β1 protein expression and their quantification. (H) mRNA levels of SYK, SH3BP2, α-SMA, TIMP1, Tgf-β1, Col1a1, FN1, Pdgfrβ , and PAI-1 . Data were presented as the mean ± SEM ( n = 4). # P < 0.05, ## P < 0.01, ### P < 0.001 versus the Tgf-β1 group; * P < 0.05, ** P < 0.01, *** P < 0.001 versus the Tgf-β1+oe-SYK group.

Article Snippet: The antibodies used are as follows: SCD1 (2794T, CST, 1:1,000), PPAR-γ (66936-1-Ig, Proteintech, 1:2,000), IL-6 (12242T, CST, 1:1,000), TLR4 (66350-1-Ig, Proteintech, 1:3,000), TIMP1 (33502-1, SAB, 1:3,000), α-SMA (67735, Proteintech , 1:20,000), Tgf-β1 (26155, Proteintech, 1:1,000), α-tubulin (66031, Proteintech, 1:20,000), GAPDH (60004-1-IG, Proteintech, 1:6,000), Phospho-Syk (2710T, CST, 1:1,000), Syk (2712T, CST, 1:1,000), SH3BP2 (PA5-87739, Invitrogen, 1:1,000), Goat anti-Mouse IgG Secondary Antibody HRP (L3032, SAB, 1:5,000), and Goat anti-Rabbit IgG Secondary Antibody HRP (L3012, SAB, 1:5,000).

Techniques: In Vitro, Western Blot, Expressing