Journal: bioRxiv

Article Title: Structural and mechanistic insights into disease-associated endolysosomal exonucleases PLD3 and PLD4

doi: 10.1101/2023.11.20.567917

Figure Lengend Snippet: (A) Analysis of activity of selected PLD3 and PLD4 missense mutant proteins by fluorophore-quencher assay. (B) Cell-based stimulation assay for selected PLD3 and PLD4 mutants. HEK293Blue TM hTLR9 cells lacking PLD3 KO were reconstituted with different doses of the indicated PLD3 and PLD4 variants and stimulated with either 2006PD, 2006PS, or 30T (control). Readout reports NFκB activation. (C-E) Effect of the indicated point mutations of PLD3 and PLD4 on protein stability. (C) Size exclusion chromatography (SEC) of wile-type and mutated PLD3 and PLD4 with His-Myc tag. The aggregation peaks are represented by the early retention fractions. (D) Effects of selected PLD3 and PLD4 mutations on observed melting temperature. (E) Detailed structures of sites of relevant point mutations. mPLD3: L306, I163 and V230 are buried inside hydrophobic pockets of the protein; modeling of I163M and V230M mutants shows increased steric hindrance. (F) Location of PLD4 R235Q. Salt bridges are represented by black dashed lines. See also Figure S6 and Table S2.

Article Snippet: HEK293Blue TM hTLR9 cell line was purchased from InvivoGen and cultured according to manufacturer’s instructions.

Techniques: Activity Assay, Mutagenesis, Cell Stimulation, Activation Assay, Size-exclusion Chromatography

Journal: Frontiers in Immunology

Article Title: Neutrophil extracellular traps formation and clearance is enhanced in fever and attenuated in hypothermia

doi: 10.3389/fimmu.2023.1257422

Figure Lengend Snippet: Pro- and anti-inflammatory cytokines release in response to infectious and sterile stimuli at all temperatures (A-K) Plasma concentration of IL-6, TNF-α, IL-1β, IL-8, IL-10, IL-17A, IL-18, IL-23, MCP-1, IFN-α2 and IFN-γ in response to ex vivo stimulation of blood by E. coli , S. aureus and mitochondria at all temperatures. The data presented are means ± SD of 4 independent experiments. *p < 0.05, **/## p < 0.01, ***/### p < 0.001; # is difference vs . respective treatment at 37°C; two-factor ANOVA followed by Dunnett’s multiple comparisons test.

Article Snippet: Human Toll-like receptor 9 (TLR9)-expressing HEK-Dual™ hTLR9 (NF/IL8) reporter cells (hkd-htlr9ni, Invivogen, San Diego, CA, USA) were selected with antibiotics according to manufacturer’s instructions and cultured in DMEM (11965092, Paisley, UK), supplemented with heat inactivated 10% (v/v) FBS (P30-3306, PAN-Biotech, Aidenbach, Germany) at 37°C in 5% CO 2 .

Techniques: Sterility, Concentration Assay, Ex Vivo

Journal: The EMBO Journal

Article Title: Secretion of mitochondrial DNA via exosomes promotes inflammation in Behçet's syndrome

doi: 10.15252/embj.2022112573

Figure Lengend Snippet: A Establishment of NLRP3‐KO‐THP‐1 cells. NLRP3‐KO‐THP‐1 cells were generated using the CRISPR/Cas9 system. Expression of NLRP3 was assessed using western blotting with an anti‐NLRP3 antibody (upper). IL‐1β production by parental WT‐ and NLRP3‐KO‐THP‐1 cells in response to LPS, ATP, MSU, and poly:dAdT (lower). B Establishment of TLR9‐expressing THP‐1 cells. Human TLR9 was introduced into THP‐1 cells by lentiviral transduction (TLR9‐Ex). Expression of TLR9 was assessed using western blotting with an anti‐TLR9 antibody (upper). TNF‐α production induced by TLR9 ligands, such as K3 and ODN2006, was elevated in TLR9‐Ex THP‐1 cells (lower). C Assessment of cGAS–STING involvement in 100K‐EV‐induced cytokine production. 100K‐EVs isolated from CS of WT THP1 cells undergoing pyroptosis were added to WT or STING‐KO THP1 cells. The level of IL‐1β (left), IL‐23 (middle), and IFN‐I (right) was quantified using the specific reporter cells for these cytokines. D Exosomes and microvesicles in 100K‐EVs. 100K‐EVs were isolated from CS of WT and Casp1‐KO THP1 cells undergoing pyroptosis, the level of exosomes and microvesicles were evaluated by Western blotting (left) using anti‐CD9 (middle) and anti‐Na + /K + ATPase (right) antibodies. E, F Inflammation caused by 100K‐EVs of Casp1‐KO THP1 cells. 100K‐EVs isolated from CS of WT and Casp1‐KO THP1 cells undergoing pyroptosis were injected into the ankle of WT mice (E) and added to WT THP1 cells (F). The evaluation of ankle swelling (upper) and histological analysis (lower) was performed after 18 h. Scale bar, 1 mm. Quantitative data from the swollen ankle are shown on the right (E). IL‐1β bioactivity in CS was measured using reporter cells for IL‐1β (F). Data information: Statistical analyses were performed using a Mann–Whitney U test (D), (median; minimum and maximum value excluding population outliers; ** P < 0.01; NS, not statistically significant), an ANOVA with Tukey's post‐hoc test (C, F) (mean ± SD; * P < 0.05, ** P < 0.01; NS, not statistically significant), or a Steel–Dwass test (E) (median; 25 th and 75 th percentile; minimum and maximum value excluding population outliers; * P < 0.05; NS, not statistically significant). For panel A and B, mean ± SD. The data are representative of two (A, B, D–F) and three (C) independent experiments. Source data are available online for this figure.

Article Snippet: HEK‐Blue TLR9 , InvivoGen , hkb‐htlr9 , .

Techniques: Generated, CRISPR, Expressing, Western Blot, Transduction, Isolation, Injection, MANN-WHITNEY

Journal: The EMBO Journal

Article Title: Secretion of mitochondrial DNA via exosomes promotes inflammation in Behçet's syndrome

doi: 10.15252/embj.2022112573

Figure Lengend Snippet:

Article Snippet: HEK‐Blue TLR9 , InvivoGen , hkb‐htlr9 , .

Techniques: Western Blot, Purification, Produced, Affinity Purification, Flow Cytometry, Confocal Microscopy, Electron Microscopy, Amplification, Recombinant, Blocking Assay, Adjuvant, Enzyme-linked Immunosorbent Assay, LDH Cytotoxicity Assay, Isolation, DNA Extraction, dsDNA Assay, Real-time Polymerase Chain Reaction, Plasmid Preparation, Clone Assay, Mutagenesis, Sequencing, Transfection, DNA Ligation, Fractionation