Fig. 4

NF-κB pathway and NLRP3 inflammasome components in S protein stimulated monocytes. Enriched monocytes from healthy donor were cultured under control conditions or stimulated with S protein 15 nM (S15). A NF-κB mRNA expression analysis by qPCR in enriched monocytes cultured for 16 h (n = 12). B Western blot protein values of phosphorylated NF-κB p65 particle (pp65) relative to β-actin protein values from lysates of enriched monocytes cultured for 16 hours (n = 3). C mRNA expression analysis by qPCR of TNF-α (n = 12) and IL-6 (n = 9) in enriched monocytes cultured for 3 hours. D IL-6 concentration in supernatant from enriched monocytes cultured for 16 hours (n = 12). 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 from enriched monocyte’s lysates cultured for 16 hours (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 from enriched monocytes cultured for 16 hours (n = 20). H Supernatant IL-1β concentration measured by CBA (n = 12). I Normalized TF to β-actin protein ratio determined by Western blot from enriched monocytes lysates cultured for 16 hours (n = 5). Differences were analyzed using Wilcoxon’s paired test. J Enriched monocytes were stimulated with S protein or control, treated or not with MCC950 and cultured for 16 h. Left: CD14⁺ gated-cells normalized mean fluorescent intensity (MFI) of NLRP3 (n = 12); center: normalized amount of Casp-1⁺ CD14⁺ cells analyzed by flow cytometry (n = 15); right: supernatant IL-1β concentration measured by CBA (n = 6). Differences were analyzed by repeated measures ANOVA and Tukey’s multiple comparison test. All data are represented as mean ± Standard Error of the Mean (SEM). Only statistically significant differences are stated: *p < 0.05, **p < 0.01., ***p < 0.001 and ****p < 0.0001