soluble mouse tlr4 (mtlr4  (Bio-Techne corporation)

Journal: bioRxiv

Article Title: Cisplatin toxicity is mediated by direct binding to TLR4 through a mechanism that is distinct from metal allergens

doi: 10.1101/2022.07.06.498982

Figure Lengend Snippet: (A) IL-8 secretion of HEK293-hTLR4 cells pre-treated with 0.1nM soluble recombinant human TLR4 and subsequently treated with either 1 ng/mL LPS, 200 μM Ni 2+ , or 25 μM cisplatin. Data are shown as the percentage of TLR4 activity in the absence of soluble hTLR4. Nil representative of cells treated without agonists ( n = 4 independent biological replicates for all conditions). (B) IL-8 secretion of HEK293-hTLR4 cells pre-treated with 0.1 nM soluble recombinant mouse TLR4 and subsequently treated with either 1 ng/mL LPS, 200 μM Ni 2+ , or 25 μM cisplatin. Data are shown as the percentage of TLR4 activity in the absence of soluble mTLR4 ( n = 4 independent biological replicates for LPS & Ni 2+ conditions, n = 8 independent biological replicates for Nil & Cisplatin conditions). Nil representative of cells treated without agonists. (C) IL-8 secretion of HEK293-hTLR4 cells pre-treated with different concentrations of soluble recombinant hTLR4 after treatment with 25 μM cisplatin as a percentage of IL-8 secretion without the pre-treatment condition ( n = 4 independent biological replicates). Data Information: For all panels, actual individual data from each experiment are plotted as box (25 th and 75 th percentile borders; median central band) with Tukey whiskers. Statistical analyses were performed through 2-way ANOVA and the Bonferroni multiple testing correction. ****, P < 0.0001; ns, not significant. For panel C, non-linear best-fit-curve follows four parameters, variable slope; R 2 = 0.59. Reported IC 50 = 0.09813 nM.

Article Snippet: Either soluble mTLR4 (Biotechne R&D Systems, Cat #9149-TR-050) or hTLR4 (Biotechne R&D Systems, Cat #1478-TR-050), were added 24 hrs post-seeding for 1 hr prior to agonist treatments.

Techniques: Recombinant, Activity Assay

Journal: bioRxiv

Article Title: Cisplatin toxicity is mediated by direct binding to TLR4 through a mechanism that is distinct from metal allergens

doi: 10.1101/2022.07.06.498982

Figure Lengend Snippet: (A-B) Microscale thermophoresis analysis showing normalized fluorescence of mTLR4 plotted against the indicated concentrations of (A) Ni 2+ ( n = 3 independent replicates) or (B) cisplatin ( n = 4 independent replicates), respectively. Data Information: (A) Apparent K d unavailable/undetectable. (B) Apparent K d = 133.6 μM; R 2 = 0.781.

Article Snippet: Either soluble mTLR4 (Biotechne R&D Systems, Cat #9149-TR-050) or hTLR4 (Biotechne R&D Systems, Cat #1478-TR-050), were added 24 hrs post-seeding for 1 hr prior to agonist treatments.

Techniques: Microscale Thermophoresis, Fluorescence

Journal: bioRxiv

Article Title: Cisplatin toxicity is mediated by direct binding to TLR4 through a mechanism that is distinct from metal allergens

doi: 10.1101/2022.07.06.498982

Figure Lengend Snippet: (Left panel) LPS requires MD-2 for TLR4 activation and cannot directly bind soluble TLR4 species. (Middle panel) Nickel can directly bind human TLR4 and this requires His456/His458. Soluble hTLR4 can block nickel activation of TLR4 (denoted by black line). Nickel cannot bind to mouse TLR4, which lacks these histidine residues. (Right panel) Cisplatin can directly bind both human and mouse TLR4 and this is not strictly dependent on His456/His458. Cisplatin has higher affinity for soluble hTLR4, which is more effective at blocking cisplatin activation of TLR4, compared to soluble mTLR4 (denoted by solid black vs. grey dashed lines). The additional TLR4 residues that contribute to cisplatin binding remain to be elucidated (denoted by ?).

Article Snippet: Either soluble mTLR4 (Biotechne R&D Systems, Cat #9149-TR-050) or hTLR4 (Biotechne R&D Systems, Cat #1478-TR-050), were added 24 hrs post-seeding for 1 hr prior to agonist treatments.

Techniques: Activation Assay, Blocking Assay, Binding Assay