Journal: Frontiers in immunology

Article Title: 1-Palmitoyl-2-Linoleoyl-3-Acetyl-rac-Glycerol (PLAG) Rapidly Resolves LPS-Induced Acute Lung Injury Through the Effective Control of Neutrophil Recruitment.

doi: 10.3389/fimmu.2019.02177

Figure Lengend Snippet: FIGURE 4 | PLAG accelerates the LPS-induced TRIF-dependent endocytosis pathway. Schematic diagram of TLR4-mediated signaling in response to LPS and PLAG/LPS in macrophages. TLR4 endocytosis by LPS treatment occurs and MIP-2 is produced by internalization signaling of TLR4, and neutrophils enter the alveoli. In the macrophages treated with PLAG, TLR4 intercellular trafficking by LPS is promoted and the internalization signaling of TLR4 is terminated rapidly, resulting in decreased expression of MIP-2, and reduction of neutrophil infiltration (A). Raw264.7 cells were transfected with siRNA targeting TRIF, TRAM, TIRAP, and Myd88, as well as the scrambled control. After 24 h, target mRNA levels were assessed by RT-PCR (B). Transfected cells were then incubated with 10 or 100 µg/ml of PLAG or DMSO (as solvent control) for 1 h and then stimulated with 100 ng/ml LPS for 16 h. Culture supernatants were assayed by ELISA to measure the secreted levels of MIP-2, IFN-β, IL-1β, and TNF (C). Raw264.7 cells were incubated with 100 µg/ml of PLAG or DMSO (as solvent control) for 1 h and then stimulated with 100 ng/ml LPS for 15, 30, 60, and 120 min. Following immunoprecipitation with TLR4/MD2 antibodies, cell lysates were separated by SDS-PAGE and analyzed by immunoblot analysis with antibodies to TRIF, Myd88, and TLR4 (D). The whole lysates were analyzed by immunoblot analysis to assess phosphorylation of TRAM or TIRAP (E) and IRF3 or p65 (F). Total mRNA was extracted from whole cells, and expression of MIP-2, IFN-β, IL-1β, and TNF was assessed by RT-PCR (G). GAPDH used as loading control. All Data represent one experiment performed in triplicate; *indicates p < 0.05.

Article Snippet: Proteins in gels were transferred onto polyvinylidine (PVDF) membranes (Bio-Rad), and these were blocked with 5% BSA (Gibco) in PBS-T. Membranes were then incubated with antibodies against TRIF (Abcam), Myd88 (Cell Signaling Technology), TLR4 (Invitrogen), phosphoTRAM (MyBioSource, San Diego, CA, USA), TRAM (R&D Systems), phospho-TIRAP (Y86; Abcam), TIRAP (Abcam), phospho-IRF3 (Ser396; Cell Signaling Technology), IRF3 (Cell Signaling Technology), phospho-p65 (Ser536; Cell Signaling Technology), and p65 (Enzo Life Sciences, Inc.) overnight at 4◦C.

Techniques: Produced, Expressing, Transfection, Control, Reverse Transcription Polymerase Chain Reaction, Incubation, Solvent, Enzyme-linked Immunosorbent Assay, Immunoprecipitation, SDS Page, Western Blot, Phospho-proteomics

Journal: Frontiers in immunology

Article Title: 1-Palmitoyl-2-Linoleoyl-3-Acetyl-rac-Glycerol (PLAG) Rapidly Resolves LPS-Induced Acute Lung Injury Through the Effective Control of Neutrophil Recruitment.

doi: 10.3389/fimmu.2019.02177

Figure Lengend Snippet: FIGURE 8 | PLAG prevents ALI through reduction of neutrophil transmigration via acceleration of TLR4 trafficking. A serial cascade is expected to occur following LPS treatment, leading to the recruitment of neutrophils into the alveolar. (1) Exogenously injected LPS binds to MD2/TLR4 on the surface of macrophage in the alveoli, resulting in activation of macrophage. (2) Activated TLR4 binds to endocytosis-dependent adaptor protein such as TRAM and TRIF, and internalized into intracellular. Then, IRF3, which is a downstream of TLR4/TRAM/TRIF complex, is activated and acts as a transcription factor in the nucleus, (3a) expressing mRNA of MIP-2 and induces the production of MIP-2 protein. MIP-2 produced from macrophage forms a concentration gradient to recruit neutrophils into alveoli. (3b) Internalization of TLR4 also produces intracellular ROS. The generated ROS remove LPS and MIP-2 induces (4) transmigration and (5) recruitment of neutrophils into alveolar. Finally, the superfluous recruited neutrophils will damage to host tissue and occur the inflammation. At this time, PLAG treatment promotes trafficking of TLR4 that occurs in response to LPS on plasma membrane of macrophage. Therefore, PLAG induces rapid removal of LPS by promoting ROS generation time and induced rapid removal of ROS. In addition, the duration of activation of IRF3 by internalization of TLR4 is reduced, which ultimately reduces the total amount of MIP-2 expression and prevents the excessive recruitment of neutrophils to the alveolar space. Upon PLAG administration, LPS-induced neutrophil transmigration is attenuated via acceleration of TLR4 trafficking.

Article Snippet: Proteins in gels were transferred onto polyvinylidine (PVDF) membranes (Bio-Rad), and these were blocked with 5% BSA (Gibco) in PBS-T. Membranes were then incubated with antibodies against TRIF (Abcam), Myd88 (Cell Signaling Technology), TLR4 (Invitrogen), phosphoTRAM (MyBioSource, San Diego, CA, USA), TRAM (R&D Systems), phospho-TIRAP (Y86; Abcam), TIRAP (Abcam), phospho-IRF3 (Ser396; Cell Signaling Technology), IRF3 (Cell Signaling Technology), phospho-p65 (Ser536; Cell Signaling Technology), and p65 (Enzo Life Sciences, Inc.) overnight at 4◦C.

Techniques: Transmigration Assay, Injection, Activation Assay, Expressing, Produced, Concentration Assay, Generated, Clinical Proteomics, Membrane

Journal: Journal of Biological Chemistry

Article Title: A potential target for liver cancer management, lysophosphatidic acid receptor 6 (LPAR6), is transcriptionally up-regulated by the NCOA3 coactivator

doi: 10.1074/jbc.ra119.009899

Figure Lengend Snippet: Fig. 5 NCOA3 regulates H3K27ac enrichment at LPAR6 locus (A) Schematic diagram of LPAR6 gene and specific primers designed for qPCR. Black box represents CDS region of LPAR6; Gray boxes represent amplification sites by ChIP-qPCR. (B-C) H3K27ac enrichment in (B) HepG2 or (C) Huh7 cells with or without NCOA3 knockdown. Equal amounts of cells were collected after NCOA3 shRNA or scrambled shRNA transfection for 48h and then subject to ChIP. Primers designed for Site 1 amplified LPAR6 coding sequence, while Site 2 and 3 amplified LPAR6 promoter. Error bars represent S.D. from three independent biological replicates. *p< 0.05 and **p< 0.01, significance of difference was analyzed by unpaired t-test. (D) HGF treatment decreased NCOA3 expression in HepG2

Article Snippet: Antibody against NCOA3 (20032-1-AP) was purchased from Proteintech.

Techniques: Amplification, ChIP-qPCR, Knockdown, shRNA, Transfection, Sequencing, Expressing

Journal: Frontiers in Pharmacology

Article Title: Achyranthes bidentata Polysaccharide Activates Nuclear Factor-Kappa B and Promotes Cytokine Production in J774A.1 Cells Through TLR4/MyD88 Signaling Pathway

doi: 10.3389/fphar.2021.753599

Figure Lengend Snippet: Primer sequences for PCR.

Article Snippet: Antibodies against Toll-like receptor 2 (TLR2), cluster of differentiation 14 (CD14), and TRIF-related adaptor molecule (TRAM) were purchased from Proteintech (IL, United States).

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Achyranthes bidentata Polysaccharide Activates Nuclear Factor-Kappa B and Promotes Cytokine Production in J774A.1 Cells Through TLR4/MyD88 Signaling Pathway

doi: 10.3389/fphar.2021.753599

Figure Lengend Snippet: ABPS increased the expression of TLR4 and MyD88 in J774 A.1 cells. (A) The mRNA expression of TLR2, TLR4, TRAM, and MyD88. (B) The protein expression of TLR2, TLR4, TRAM, and MyD88. The blot shown is representative of one of the three similar experiments. Cells were treated 500 μg/ml ABPS for 24 h. NC: normal control; ABPS500: 500 μg/ml ABPS treated. * p < 0.05, ** p < 0.01 vs. NC. The values are presented as means ± SD.

Article Snippet: Antibodies against Toll-like receptor 2 (TLR2), cluster of differentiation 14 (CD14), and TRIF-related adaptor molecule (TRAM) were purchased from Proteintech (IL, United States).

Techniques: Expressing