Kinase experiment:

HEK293T cells are transfected with an NF-κB-dependent luciferase reporter plasmid and clones with stable expression are selected by culture in DMEM containing puromycin. Cells are cotransfected with constructs for mouse or human TLR4 plus mouse or human MD-2, and 2 d later are stimulated with 50 μM Neoseptin-3 or 1 μg/mL LPS for 6 h. Cells are lysed, and luciferase activity is measured[1].

Cell experiment:

Cells are seeded onto 96-well plates at 1×105 cells per well and stimulated with Neoseptin-3 [dissolved in DMSO; final DMSO concentrations (≤0.2%) are kept constant in all experiments] for 4 h. Mouse TNFα, IL-6, or IFN-β, or human TNFα in the supernatants are measured by ELISA kits according to the manufacturer’s instructions. Unless otherwise indicated, mouse cells are from wild-type C57BL/6J mice[1].

Neoseptin 3 is a Toll-like receptor 4/myeloid differentiation factor 2 (mTLR4/MD-2) agonist with an EC50 of 18.5 μM.

Neoseptin 3 is a Toll-like receptor 4/myeloid differentiation factor 2 (mTLR4/MD-2) agonist with an EC50 of 18.5 μM. Neoseptin-3 induces TNFα production by macrophages in a concentration-dependent manner. Neoseptin-3 induces phosphorylation of IκB kinases α (IKKα), IKKβ, p38, c-Jun N-terminal kinase (JNK), and ERK, and degradation of IκBα, consistent with activation of MAPK and canonical NF-κB signaling. TANK-binding kinase 1 (TBK1) and IRF3 phosphorylation also increase in response to Neoseptin-3[1].

[1]. Wang Y, et al. TLR4/MD-2 activation by a synthetic agonist with no similarity to LPS. Proc Natl Acad Sci U S A. 2016 Feb 16;113(7):E884-93.