Journal:

Article Title: Lipopolysaccharide-Trap-Fc, a Multifunctional Agent To Battle Gram-Negative Bacteria ▿

doi: 10.1128/IAI.00004-09

Figure Lengend Snippet: LPS-Trap-Fc1 to -Fc4 bind LPS and block LPS-mediated IL-6 production in Mono Mac 6 cells. (A) Supernatants of HEK293T cells transfected with LPS-Trap-Fc1 were incubated in the presence (lane 1) or absence (lane 3) of biotin-LPS. Additionally, supernatants were preincubated with an anti-TLR4/MD-2 MAb (lane 2) or an excess of LPS (lane 4). Complexes were immunoprecipitated with streptavidin-Sepharose, subjected to SDS-PAGE, and analyzed by Western blotting with an anti-FLAG MAb. (B) RAW 264.7 cells were incubated with LPS-Trap-Fc1 or a medium control. Cells were stimulated with 100 ng/ml LPS overnight, and IL-6 levels in supernatants were determined by ELISA. *, P < 0.002 compared to control (Mann-Whitney U test, n = 6).

Article Snippet: For specificity control, some supernatants were preincubated with either 50 μg/ml LPS ( Salmonella enterica serovar Minnesota; Sigma), which has a high binding activity for TLR4/MD-2, or 10 μg/ml anti-mouse TLR4/MD-2 MTS510 MAb (Hycult Biotechnology, Uden, The Netherlands) for 1 h at room temperature.

Techniques: Blocking Assay, Transfection, Incubation, Immunoprecipitation, SDS Page, Western Blot, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

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: European Journal of Inflammation

Article Title: Mechanism of lipopolysaccharide-mediated induction of epithelial-mesenchymal transition of alveolar type II epithelial cells in absence of other inflammatory cells

doi: 10.1177/20587392211014427

Figure Lengend Snippet: Figure 1. Temporal changes in mRNA expression levels of EMT-related markers in A549 cells upon LPS treatment. LPS (net concentration 10 µg/mL) was added to A549 cells, and real-time PCR was used to measure the mRNA levels of EMT-related markers 0.5, 1, 2, 3, 6 and 12 h after LPS treatment. The epithelial marker E-cadherin mRNA decreased significantly compared to the untreated control group at each time point (a), while the mesenchymal marker vimentin mRNA increased significantly 2 h after LPS stimulation (b). The expression of TLR4 mRNA increased 1 h after LPS treatment (c) and that of TGF-β1 mRNA significantly increased 1, 2, 6 and 12 h post LPS treatment (d). Smad2/3 mRNA expression also significantly increased 1 h after LPS treatment (e). Error bars represent mean ± standard deviation, and * indicates a significant difference compared to the control group (p < 0.05) (n = 3).

Article Snippet: To further investigate whether the TLR4 pathway played a role in EMT, we pharmacologically inhibited TLR4 in A549 cells, using the small molecule TAK242 (10 μM, Selleck Chemicals, America), for 0.5 h prior to LPS treatment for 1 h and examined corresponding effects on EMT.

Techniques: Expressing, Concentration Assay, Real-time Polymerase Chain Reaction, Marker, Control, Standard Deviation

Journal: European Journal of Inflammation

Article Title: Mechanism of lipopolysaccharide-mediated induction of epithelial-mesenchymal transition of alveolar type II epithelial cells in absence of other inflammatory cells

doi: 10.1177/20587392211014427

Figure Lengend Snippet: Figure 4. TGF-β1 expression level upon pharmacological inhibition of TLR4 prior to LPS stimulation. A549 cells were pretreated with TAK242 (10 µM) for 0.5 h before stimulating with LPS (10 µg/mL) for 1 h; the levels of TGF-β1 mRNA were significantly reduced compared with those in the untreated control group. Error bars represent mean ± standard deviation, and * indicates a significant difference compared to the control group (p < 0.05) (n = 3).

Article Snippet: To further investigate whether the TLR4 pathway played a role in EMT, we pharmacologically inhibited TLR4 in A549 cells, using the small molecule TAK242 (10 μM, Selleck Chemicals, America), for 0.5 h prior to LPS treatment for 1 h and examined corresponding effects on EMT.

Techniques: Expressing, Inhibition, Control, Standard Deviation

Journal: European Journal of Inflammation

Article Title: Mechanism of lipopolysaccharide-mediated induction of epithelial-mesenchymal transition of alveolar type II epithelial cells in absence of other inflammatory cells

doi: 10.1177/20587392211014427

Figure Lengend Snippet: Figure 3. Quantification of changes in vimentin protein level after inhibition of TGF-β1 and TLR4 prior to stimulation by LPS. A549 cells were treated with LPS (10 µg/mL) with or without pretreatment with the TLR4 inhibitor TAK242 (10 µM) or the TGF- β1 inhibitor EW7197 (10 µM) for 0.5 h. Pretreatment of A549 cells with inhibitors targeting TLR4 (a, c) and TGF-β1 (b, d) before LPS stimulation significantly lowered the expression of vimentin. Error bars represent mean ± standard deviation, and * indicates a significant difference between pretreatment group and LPS group (p < 0.05) (n = 3).

Article Snippet: To further investigate whether the TLR4 pathway played a role in EMT, we pharmacologically inhibited TLR4 in A549 cells, using the small molecule TAK242 (10 μM, Selleck Chemicals, America), for 0.5 h prior to LPS treatment for 1 h and examined corresponding effects on EMT.

Techniques: Inhibition, Expressing, Standard Deviation