Psirna htlr4

Human acute monocytic leukaemia THP‐1 cells were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured in RPMI 1640 medium, supplemented with 10% foetal bovine serum (Thermo Fisher Scientific) at 37°C and in a 5% CO₂ atmosphere. Fresh whole blood containing the anticoagulant heparin was mixed with phosphate‐buffered saline (PBS) at a ratio of 1:1 and then added gently into an equal volume of Ficoll‐Paque Premium (STEMCELL Technologies, Vancouver, BC, Canada). After centrifugation at 1000 g for 30 minutes, PBMCs were gently washed with PBS twice before discarding the supernatant. Then, the PBMCs were resuspended with RPMI 1640 medium for further use. A hemocytometer was used to count the cells.
RNA interference was performed to silence the TLR2 and TLR4 genes (psiRNA‐hTLR2 and psiRNA‐hTLR4; InvivoGen, San Diego, CA, USA). THP‐1 cells (1 × 10⁴) were plated in 96‐well plates and pretreated with 0.2 μg of psiRNA and Lipofectamine 2000 (Thermo Fisher Scientific) for 24 hours. In addition, THP‐1 cells (1 × 10⁵) were also plated into 6‐well plates and pretreated with 2.0 μg of psiRNA and Lipofectamine 2000 (Thermo Fisher Scientific) for 24 hours.

The human monocytic cell line (purchased from CTCC, Wuhan University, Wuhan, China) was cultured as previously described (Xie et al., 2017 (link)). psiRNA-hTLR2, psiRNA-hTLR4, psiRNA-hTLR6, pZERO-hTLR2, pZERO-hTLR4, pZERO-hTLR6, pDeNy-hMyD88, pDeNy-hMal, and control plasmids (psiRNA-LucGL3, pZERO-mcs, and pDeNy-mcs) were obtained from InvivoGen (Carlsbad, United States). The inhibitor of NF-κB, BAY11-7082, was obtained from Cell Signaling Technology (Beverly, MA, United States). Anti-phosphorylated and anti-total IκBα and p65 mouse antibodies were obtained from Abcam (Cambridge, MA, United States). Anti-β-actin antibody was bought from Sigma-Aldrich (St, Louis, MO, United States). Cy3-conjugated goat anti-mouse IgG antibody was obtained from Invitrogen (Frederick, MD). Antibodies against TLR2, TLR4, TLR6, MyD88, and Mal were purchased from Abcam (Cambridge, MA, United States). Neutralizing antibodies against TLR2, TLR4, and TLR6 were obtained from Novus. Complete protease inhibitor cocktail was purchased from Roche Applied Science (Mannheim, Germany). Other reagents were purchased from Thermo Fisher Scientific (Waltham, United States).