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: Immunology and cell biology

Article Title: Calcineurin B stimulates cytokine production through a CD14-independent Toll-like receptor 4 pathway.

doi: 10.1038/icb.2015.91

Figure Lengend Snippet: Figure 2 The CnB–TLR4 interaction results in cytokine production through IκB-α phosphorylation. (a), Quantitative PCR for NF-κB (2.1-fold) and IκB-α (9.3- fold). Po0.001, N = 3. (b) Western blot to detect IκB-α (with or without phosphorylation) and IL-8 in CnB-treated (lane 2) or buffer solution-treated (lane 1) U937 cells; the cells were stimulated with CnB or its buffer solution for 3 h. (c) Western blot to detect IκB-α phosphorylation at various time points in CnB- treated U937 cells. (d) Western blot to detect IκB-α phosphorylation in U937 cells treated with different antibodies. (e and f) ELISA for IL-8 and TNF-α in HEK293 cells transfected with TLR4 and/or MD2 and treated with CnB and LPS for 3 h. The error bars represent the mean ± s.d.

Article Snippet: A recombinant plasmid containing TLR4 and/or MD2 (Addgene, Cambridge, MA, USA) was transfected into HEK293s using the Lipofectamine 3000 Transfection Reagent (Invitrogen), and then selected with neomycin for 6 days.

Techniques: Phospho-proteomics, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Transfection

Journal: Scientific Reports

Article Title: Pseudomonas aeruginosa -derived DnaJ functions as a novel immunomodulator inducing IFNβ via CME–SGK1–IRF3 axis in macrophages

doi: 10.1038/s41598-025-31281-x

Figure Lengend Snippet: DnaJ-induced IFNβ expression is regulated by TLR4. ( A , B , D , F – H , K , L ) dTHP-1 cells were pretreated for 1 h with: ( A , B ) IRAK4 IH (compound 26), ( D ) TLR2/4 IH (OxPAPC), ( F – H ) TLR4 IH (CLI-095; 3 µM in H ), or ( K , L ) MyD88 IH (T6167923). ( C , E , I , J ) Cells were transfected with: ( C ) siTLR10, ( E ) siTLR2, ( I ) siCD14, or ( J ) siMD2 for 48 h. Knockdown efficiency is shown in the right panel of ( C , E , I , J ). Following pretreatment or transfection, cells were treated with DnaJ (1 µg/ml) for 4 h ( A – G , I – L ) or for the indicated durations ( H ). IFNβ mRNA was quantified by qPCR; protein levels were analyzed by immunoblotting. The samples used for immunoblotting were obtained from the same experiment, and the original blots/gels are shown in the Supplementary Figure. Data in ( A – G , I – L ) are expressed as means ± SD ( n = 3). Immunoblots are representative of three experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001 vs. DnaJ treatment alone. Inhibitor (IH).

Article Snippet: IRF3 siRNA (siIRF3; #sc-35710), siTLR2 (#sc-40256), siCD14 (#sc-29248), and siMD2 (#sc-35889) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). siTLR10, siTRIF, and siSGK1 were purchased from Bioneer (Daejeon-si, Korea). dTHP-1 macrophage cells (5 × 10 5 cells) were transfected with siRNA or a negative control siRNA-A (#sc-37007) at 20–200 nM using Lipofectamine RNAiMax (Invitrogen).

Techniques: Expressing, Transfection, Knockdown, Western Blot

Journal: Advanced Science

Article Title: Megasphaera Elesdenii Dysregulates Colon Epithelial Homeostasis, Aggravates Colitis‐Associated Tumorigenesis

doi: 10.1002/advs.202505670

Figure Lengend Snippet: M. elsdenii ‐mediated DC activation depends on TLR4/NF‐κB/IRF4 pathway. A) Volcano plot of RNA sequencing dataset showing differential expressed genes (DEGs) of BMDCs infected with M. elsdenii compared with PBS treated BMDCs ( p <0.05, fold‐change [FC] R 2.0) derived from biological triplicates. Red: upregulated, blue: downregulated. B) Gene set enrichment analysis (GSEA) showing the enrichment of LPS‐treatment signature. C) Colonic tissue supernatants and serum LPS levels were determined using ELISA in M. elsdenii ‐treated SPF mice (top) or ABX‐mice (bottom) (n = 5/group). D) GSEA showing the enrichment of IRF4‐high signature. E) Flow cytometric analysis of CCR7, CD80 and CD86 expression in M. elsdenii ‐treated BMDCs with and without TLR2/4 antagonist (n = 5 independent experiments). F) Western blot analysis of key proteins of TLR4/NF‐κB/IRF4 pathway in TLR2/4 antagonist treated BMDCs. G) Pull‐down analysis of BMDC lysates incubated with biotin‐labeled LPS derived from M. elsdenii or E. coli . H) qPCR analysis of Irf4 mRNA levels (top) and western blot analysis of IRF4 protein levels (bottom) in BMDCs transfected with IRF4 shRNA or vector (n = 5 independent experiments). I) Flow cytometric analysis of CCR7, CD80 and CD86 expressions in M. elsdenii or PBS treated BMDCs transfected with IRF4 shRNA or vector (n = 5 independent experiments). Each experiment conducted at least three replicates. Data presented as mean ± SEM. Statistical analysis was performed with Student's t test C, H), one‐way ANOVA E, I); ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: In indicated experiments, M. elsdenii ‐infected BMDCs were treated with selected TLR2 antagonist (TLR2‐IN‐C29, Selleck) or TLR4 antagonist (MD2‐IN‐1, Selleck) at a concentration of 10 × 10 −6 m . To isolate spleen CD4+ T cells, spleen was extracted and mechanically minced and pressed using syringe plungers through a 70 mm cell strainer.

Techniques: Activation Assay, RNA Sequencing, Infection, Derivative Assay, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Incubation, Labeling, Transfection, shRNA, Plasmid Preparation

Journal: Advanced Science

Article Title: Megasphaera Elesdenii Dysregulates Colon Epithelial Homeostasis, Aggravates Colitis‐Associated Tumorigenesis

doi: 10.1002/advs.202505670

Figure Lengend Snippet: M. elsdenii has no impact on inflammation‐related tumorigenesis in Tlr4 ‐deficient mice. A) Schematic of in vivo experiment design of M. elsdenii ‐colonized WT and Tlr4 −/− AOM/DSS model. B) DAI score of mice (n = 7–8/group). C) Representative macroscopic view of the colons and polyps in M. elsdenii ‐colonized WT and Tlr4 −/− AOM/DSS mice. D) Polyp numbers (left) and size (right) in M. elsdenii ‐colonized WT and Tlr4 −/− AOM/DSS mice (n = 5/group). E) Representative H&E images of colonic tissue sections (Left, scale bar = 1 mm) and histological scoring of colonic inflammatory pathology and dysplasia of tumors (right). F, G) Flow cytometric analysis of CD45+ hematopoietic cells, CD11c+MHCII+ DCs, and CD4+ T cells frequency in colonic LP of M. elsdenii ‐colonized WT and Tlr4 −/− AOM/DSS mice (n = 4/group). H) qPCR analysis of Th1‐ and Th17‐associated transcription factors ( Tbx21 and Rorc ) and cytokines ( Ifng and Il17a ) in the colon tissue ( n = 4/group). I) Flow cytometric analysis of CCR7, CD80, and CD86 expression in Tlr4 ‐deficient or WT BMDCs ( n = 5 independent experiments). Each experiment conducted at least three replicates. Data presented as mean ± SEM. Statistical analysis was performed with two‐way ANOVA B), one‐way ANOVA (D, E, G‐I); * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: In indicated experiments, M. elsdenii ‐infected BMDCs were treated with selected TLR2 antagonist (TLR2‐IN‐C29, Selleck) or TLR4 antagonist (MD2‐IN‐1, Selleck) at a concentration of 10 × 10 −6 m . To isolate spleen CD4+ T cells, spleen was extracted and mechanically minced and pressed using syringe plungers through a 70 mm cell strainer.

Techniques: In Vivo, Expressing