Psirna lucgl3

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).

The cells were seeded at a density of 2 × 10⁵ cells in 6‐well plates. Transfection of hMSCs was performed using psiRNA‐hTLR4 or psiRNA‐LucGL3 (control plasmid) (InvivoGen, Catalog No. ksirna42‐htlr4), and the lipofectamine 3000 (Invitrogen, Catalog No. L3000075) reagent according to the manufacturer's instructions. siRNA‐induced silencing of TLR4 gene was determined by Real‐time PCR and western blot techniques.