Fig. 5

Effect of S protein stimulation over monocytes treated with TLR4 inhibitor TAK242. (A-I) Enriched monocytes from healthy donor were cultured for 16 h and were stimulated or not with S protein (S15) and treated or not with TLR4 inhibitor (TAK242). A NF-κB mRNA expression analysis by qPCR. (n = 9). B Western blot protein values of phosphorylated NF-κB p65 particle (pp65) relative to β-actin protein values (n = 3). C mRNA expression analysis by qPCR of TNF-α (n = 10) and IL-6 (n = 7). D IL-6 concentration in supernatant from enriched monocytes (n = 10). E Left: CD14⁺ gated-cells normalized mean fluorescent intensity (MFI) of NLRP3 (n = 20); right: normalized NLRP3 to β-actin protein ratio determined by Western blot (n = 7). F CD14⁺ gated cells normalized mean fluorescent intensity (MFI) of ASC (n = 12). G Normalized amount of active caspase positive (Casp-1⁺) CD14⁺ cells analyzed by flow cytometry (=20). H Supernatant IL-1β concentration measured by CBA (n = 12). I Normalized TF to β-actin protein ratio determined by Western blot (n = 5). Differences were analyzed by repeated measures ANOVA and Tukey’s multiple comparison test. J-K Enriched monocytes from healthy donor were transfected in absence (control) or presence of TLR4 siRNA (siTLR4) and cultured for 16 h with S15 or not. J Normalized amount of CD14⁺ cells expressing surface TLR4 (n = 7). K Left: CD14⁺ gated-cells normalized mean fluorescent intensity (MFI) of NLRP3 (n = 7): right: normalized amount of active casp-1⁺ CD14⁺ cells analyzed by flow cytometry (n = 7). Differences were analyzed using Wilcoxon’s paired test. All data are represented as mean ± SEM. Only statistically significant differences are stated: *p < 0.05, **p < 0.01, and ***p < 0.001