Bay 11 7082

For inhibition experiments mDCs were pre-incubated with the indicated amounts of rapamycin (mTOR inhibitor), BAY-11-7082 (irreversible inhibitor of TNF-α-induced IkB-α phosphorylation resulting in inactivation of NFkB), triptolide (NFκB inhibitor), dexamethason (NFκB and MAPK inhibitor), the IKK-β inhibitors TPCA-1 (Abcam, Cambridge, UK) and BMS-345541 (Abcam, Cambridge, UK), as well as the MAPK inhibitors SP600125 (JNK MAPK inhibitor, Invivogen, Toulouse, France), SB-202190 (p38α/β MAPK inhibitor, Invivogen, Toulouse, France), or U0126 (MEK1/2 MAPK inhibitor, Cell Signaling Technologies, Leiden, The Netherlands) for 90 min and subsequently stimulated with equimolar amounts of rFlaA + rBet v 1 or rFlaA:Betv1 for either 30 min (Western Blot), 24 h (ELISA and cytotoxicity), or 72 h (ELISA and analysis of cell metabolic state). Toxicity of the used inhibitors was determined using the fixable viability dye eFlour780 (Thermo Fisher Scientific, Darmstadt, Germany, Repository Figure S1). Inhibitor concentrations showing toxic effects were excluded from the analysis.

Murine peritoneal exudate macrophages or human THP-1 cell-derived macrophages were stimulated with 0.1–1 μg/ml of SARS-CoV-2 spike recombinant protein S1 subunit (Arigo Biolaboratories, Hsinchu City, Taiwan), 100 ng/ml of a TLR4 agonist LPS from Escherichia coli 055:B5 (LPS-B5 Ultrapure; InvivoGen, San Diego, CA, USA), or 10 ng/ml of a TLR2 agonist Pam2CSK4 (InvivoGen) to induce pro-inflammatory responses. Cells were treated with 2 μM BAY 11-7082 (Abcam, Cambridge, UK), 10 μM SP600125 (Sigma-Aldrich), 0.1 or 1 μg/ml of LPS from Rhodobacter sphaeroides (LPS-RS Ultrapure; InvivoGen), or 5 μg/ml of anti-murine or anti-human TLR2 neutralizing antibodies (InvivoGen) to block NF-κB, c-Jun N-terminal kinase (JNK), TLR4, or TLR2 signaling, respectively. The final concentrations of vehicles (H₂O or dimethyl sulfoxide) to dissolve these agents were equivalent (less than 0.1%) in the culture medium among experimental groups.

GSPE (≥95.0%) was obtained from JF-Natural Company (Tianjin, China). BAY 11-7082 and antibodies against IKK, caspase-3, and NF-κB (p65) were supplied by Abcam (Cambridge, England), and antibodies against IκB, phospho-IκB (p-IκB), and NF-κB (p100/p50) were procured from Cell Signaling Technology Inc. (Danvers, MA). Antibodies against GAPDH were purchased from Goodhere Biotechnology (Hangzhou, China). Dulbecco's modified Eagle's medium (DMEM), penicillin, streptomycin, fetal bovine serum (FBS), and trypsin/EDTA were purchased from HyClone (Logan, Utah). 3-(4,5-Dimethylthiazol-2-yl)-3, 5-diphenyltetrazolium bromide (MTT) was obtained from Jiancheng Biotechnology Co. (Nanjing, China). The annexin V-FITC/PI apoptosis kit was procured from Multisciences (Hangzhou, China). ELISA kits for IL-6 and COX-2 were purchased from Elabscience (Wuhan, China).

The cell lines (BGC823, AGS, HGC27, SGC7901, MKN-45, and SGC-7901) were obtained from the Chinese Academy of Sciences (Shanghai, China). The cells were subcultured for less than half a year after the start of culture in our laboratory. All cell lines were maintained in a 37°C 5% CO₂ incubator. RPMI-1640 medium (HyClone, USA) was mixed with 1% penicillin-streptomycin (Solarbio, China) and 10% fetal bovine serum (Gibco, USA) to culture the cells. The treatment with TNF-α (10 ng/ml, Sigma-Aldrich, USA) was performed for 24 h. Bay 11–7082 (10 μM, Abcam, Cambridge, UK) treatment was performed for 12 h.

Human umbilical vein ECs (HUVECs) and ECs from healthy donor and patients were cultured in EGM‐2 (EBM‐2 + SingleQuots™ Kit) and 2% foetal bovine serum (FBS) (Lonza). HUVECs and ECs were used between P2 and P6 passages. HEK293T (ATCC) were grown in Dulbecco's modified Eagle's medium (DMEM) with 4.5 g/l glucose, 2 mM l‐glutamine, without Na pyruvate (Lonza), 10% FBS and 1% penicillin/streptomycin (Pen/Strep). Reagents and doses were as follows: LysoTracker (Thermo Fisher), bafilomycin A1 (Sigma, 200 nM), torin‐1 (CST, 10 μM), BAY11‐7082 (Abcam, 300 nM), TYRPHOSTIN AG1288 (Abcam, 300 nM), SB202190 (Tocris, 300 nM), cycloheximide (Sigma, 2 μM) and MG132 (Sigma, 2 μM).

Recombinant PF4, chondroitinase ABC, gelatin and Surfen were purchased from Millipore-Sigma (Oakville, ON, Canada). Antibodies against MMP-2, PF4, and chondroitin sulfate, as well as the NF-κB inhibitor BAY 11-7082, were obtained from Abcam (Cambridge, MA). The anti-GAPDH antibody was from HyTest (Turku, Finland). The anti-β-actin antibody was from Santa Cruz Biotechnologies (Dallas, TX). Heparinase III, and antibodies against phospho-NF-κB (Ser536), total NF-κB, and horseradish peroxidase-conjugated secondary antibodies were from Cell Signaling Technologies (Billerica, MA). The Alexa-Fluor-488-conjugated secondary antibody, and Alexa-Fluor-568 phalloidin were from Life Technologies (Grand Island, NY).