St2825

Monoclonal rabbit anti-SDC-1, anti-VCAM-1, anti-MyD88, anti-TLR4, anti-p-IκBα-Ser36 antibodies and monoclonal mouse anti-CD31 antibodies were purchased from Abcam (Cambridge, UK). Monoclonal rabbit anti-vascular endothelial- (VE-) cadherin and anti-NF-κB/p65 antibodies were bought from Cell Signaling Technology (Boston, MA, USA). Tetramethylrhodamine- (TRITC-) phalloidin (which binds to F-actin) was from Solarbio (Beijing, China), and 4′,6-diamidino-2-phenylindole (DAPI) was from SouthernBiotech (Birmingham, USA). Anti-HMGB1 antibody was from Wanleibio (Shenyang, China), and control IgG antibody was from Beyotime (Shanghai, China). Glycyrrhizin (GLY; an HMGB1 inhibitor) was obtained from JK Scientific (Beijing, China). TAK242 (a TLR4 inhibitor), ST2825 (a MyD88 inhibitor), and SN50 (an inhibitor of NF-κB/p65 translocation) were purchased from MedChemExpress (NJ, USA). Fluorescein isothiocyanate-labeled bovine serum albumin (FITC-BSA) was purchased from Solarbio (Beijing, China). A human SDC-1 enzyme-linked immunosorbent assay (ELISA) kit was purchased from Abcam (Cambridge, UK), and a mouse SDC-1 ELISA kit and VCAM-1 ELISA kit were purchased from Zcibio (Shanghai, China). A Total Nitric Oxide (NO) Assay Kit was purchased from Beyotime Biotechnology (Shanghai, China).

BMDMs were prepared as previously described with modifications[20 (link)]. Briefly, bone marrow cells were isolated from the leg bones of wild-type and TLR2^-/- mice and cultured in DMEM (Gibco, USA) supplemented with 10% FBS (Gibco, USA) and 50 ng/mL macrophage colony-stimulating factor (M-CSF) (Peprotech, USA) and maintained in a 5% CO₂ incubator at 37°C. Six days after the initial BM cell culture, the medium was changed, and the purity of F4/80⁺ cells was > 99%, as determined by flow cytometry.
In some experiments, BMDM cells (5 × 10⁵ cells/mL) were pretreated with one of the following inhibitors: 10 μM BAY 11–7082 (NF-κB inhibitor, Beyotime biotechnoogy, China), 10 μM SP 600125 (JNK MAPK inhibitor, Beyotime biotechnoogy, China), 1 μM SB 203580 (p38 MAPK inhibitor, Beyotime biotechnology, China), 10 μM PD 98059 (ERK MAPK inhibitor, Beyotime biotechnology, China) and 10 μM ST 2825 (MyD88 homodimerization inhibitor, MedChem Express, USA). Furthermore, LPS from Escherichia coli serotype O111:B4 (Sigma, USA) and synthetic lipoprotein Pam3CSK4 (InvivoGen, USA) were used in some experiments.

The Escherichia coli 0111: B4 LPS, N-hexanoyl-D-sphingosine (C6-ceramide) and zVAD-FMK were obtained from Sigma-Aldrich (St. Louis, MO, USA). DAPK1 inhibitor and ST2825 were purchased from Medchem Express (Monmouth Junction, New Jersey, USA). Recombinant GST-DAPK1 fusion protein was obtained from Millipore (Billerica, MA). Caspase-3 activity detection kit was from Bestbio (Shanghai, China). Quantikine human IL-6 ELISA kit was from R&D Systems (Minneapolis, MN). Annexin V-FITC apoptosis detection kit was ordered from Beyotime (Nanjing, China). The following antibodies with the company and concentration were used for coimmunoprecipitation (co-IP) or western-blotting analyses: anti-Pellino1 (Abcam, 1:500), anti-MyD88 (Cell Signaling Technology, 1:500), anti-caspase-8 (Cell Signaling Technology, 1:500), anti-TRIF (Cell Signaling Technology, 1:1000), anti-RIP1 (BD Biosciences, 1:2000), anti-Flag (ProteinTech group, 1:1000), anti-phospho-DAPK1 (Sigma-Aldrich, 1:1000), anti-DAPK1 (Cell Signaling Technology, 1:1000), anti-Fbxw7 (Abcam, 1:1000), anti-pSer (Santa Cruz, 1:1000), anti-Fn14 (Cell Signaling Technology, 1:2000) and anti-GAPDH (Biosynthesis, 1:3000).