Pam3ck

Chemical reagents, Pam3CK and lipopolysaccharide (LPS) were purchased from Sigma (Sigma, St. Louis, MO, USA). RPMI-1640, DMEM medium and FBS were obtained from Gibco (Gibco, Eggenstein, Germany). EBioscience (eBioScience, San Diego, CA, USA) was the source of mouse IL-6 ELISA Kit, mouse TNF-α ELISA Kit and anti-rhMD2 antibody. Trizol-reagent was purchased from Invitrogen (Invitrogen, Carlsbad, CA, USA). Two-step M-MLV Platinum SYBR Green qPCR SuperMix-UDG kit (Invitrogen, Carlsbad, CA). Chemiluminescent EMSA Kit, ECL detection reagent, protein A + G agarose, biotin-labeled NF-κB probe and Cellular NF-κB p65 Translocation Kit were obtained from Beyotime Biotechnology (Beyotime Biotech, Nantong, China). Anti-GAPDH, anti-IκBα, anti-p-ERK, anti-ERK, anti-CD68, anti-TLR4 and anti-MD2 antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-p-P38, anti-P38, anti-p-JNK and anti-JNK were from Cell Signaling Technology (Cell Signaling Technology, Danvers, MA, USA). Recombinant human MD2 (rhMD2) protein, Recombinant human TLR4 (rhTLR4) protein, and TLR2 inhibitor CU-CPT22 were purchased from R&D Systems, Inc. (Minneapolis, MN, USA). Mutated rhMD2 was obtained using the methods described in our previous publication.35 (link)

Compound L2H21 was provided by our laboratory and purified using HPLC with a purity of 99.3%. In in vitro experiments, L2H21 was dissolved in dimethyl sulphoxide (DMSO) solution and similar volume of DMSO followed as a vehicle control. In the in vivo mortality study, L2H21 was initially dissolved in water with macrogol 15 hydroxystearate (a non‐ionic solubilizer for injection from BASF) in water. The concentration of L2H21 and solubilizer was 2 mg/ml and 8% in water solution, respectively. For the vehicle, the solubilizer was prepared at 8% in water. In the in vivo ALI study, L2H21 was resuspended in 0.5% CMC‐Na solution. LPS, fluorescein isothiocyanate‐labelled LPS (FITC‐LPS) and Pam3CK were purchased from Sigma‐Aldrich (St. Louis, MO, USA). Anti‐CD68, anti‐MD‐2 and anti‐TLR4 antibodies were purchased from Santa Cruz (Santa Cruz, CA, USA). Recombinant human MD‐2 (rhMD‐2) protein was purchased from R&D Systems, Inc. (Minneapolis, MN, USA). Mutated rhMD‐2 protein was obtained by the methods described in our previous publication 19.