Bay11 7082

Cell migration assays were performed using 24-well Transwell plates (Corning Costar, Pittston, PA). First, HDVSMCs were pretreated with T. pallidum at different MOIs (T. pallidum: cell ratios of 1:1, 10:1, 100:1 and 200:1) at 37°C in an atmosphere containing 5% CO₂ for 24 h in 24-well culture plates. Then, 600 μL of cell culture supernatant was collected and added to the lower chambers of Transwell plates. Approximately 2.5 ×10⁵ THP-1 cells in a volume of 200 μL were seeded in the upper chambers of the Transwell plates. The Transwell plates were then incubated at 37°C in an atmosphere containing 5% CO₂ for 2 h. THP-1 cells that migrated to the lower chamber were counted under a microscope, and the migration rate of THP-1 cells was calculated as follows: migration rate of THP-1 cells (%) = (number of THP-1 cells in the lower chamber/number of THP-1 cells added to the upper chamber) ×100. For the inhibition assays, HDVSMCs were pre-incubated with an anti-MCP-1 neutralizing antibody (30 μg/mL) (R&D Systems, Inc., Minneapolis, MN, USA) or BAY11-7082 (2 μmol/L) for 1 h. Subsequently, HDVSMCs were incubated with T. pallidum at an MOI of 100:1 for 24 h. HDVSMCs treated with phosphate-buffered saline (PBS) were used as the control cells. The migration assay was performed, and the migration of THP-1 cells was calculated as described above.

Lipopolysaccharide (LPS, E. coli 0111: B4, TLR4 ligand), poly(I:C) (TLR3 ligand), and CpG ODN (TLR9 ligand) were purchased from Sigma-Aldrich. ChIP Grade Protein G Magnetic Beads, Cell Lysis Buffer, Anti-Myd88, Anti-Traf6, Anti-p65, Anti-p-p65 (Ser536), Anti-Ikkα/β, Anti-p-Ikkα/β (S176/180), Anti-IκBα, Anti-p-IκBα (S32), Anti-ERK, Anti-p-ERK (Thr202-Tyr204), Anti-JNK, Anti-p-JNK (Thr183-Tyr185), Anti-p38, Anti-p-p38 (Thr180-Tyr182) and Anti-Syk and Anti-p-Syk (Tyr-525/526) were from Cell Signaling Technology. Anti-DAP12 was purchased from Abcam. Anti-β-actin, Anti-rabbit IgG-HRP, and anti-mouse IgG-HRP were from Santa Cruz Biotechnology. Anti-LaminA/C, Anti-Flag and Anti-Myc and Anti-V5 were purchased from Proteintech. Anti-Ocilrp2 (AF3370) was from purchased R&D. The NF-κB inhibitor BAY11-7082 (10 μM), Tbk inhibitor Amlexanox (10 μM), Mek inhibitor PD98059 (10 μM), PI3K inhibitor Wortmannin (5 μM), Erk inhibitor SHC772984 (10 μM), Jnk inhibitor SP600125 (10 μM), or p38 inhibitor SB203580 (10 μM), Syk inhibitor R406 (5 μM) were from Selleckchem.