Fig. 1

S protein promotes NLRP3 priming and NF-κB activation in primary HUVEC cultures. Human umbilical vein endothelial cells (HUVEC) were treated with viral S protein at 7, 35 and 70 nM and IL-1β at 2.5 ng/mL for 18 h and the protein levels of the NLRP3 inflammasome system components A NLRP3 (n = 5), B pro-IL-1β (n = 4), C pro-casp-1 (n = 5), and D ASC protein levels (n = 3) were analyzed in total cell lysates by Western blot. In addition, NF-κB activation was quantified by means of E phospho-p65 (P-p65) protein levels by Western blot (n = 3) and F visualized by indirect immunofluorescence as the translocation of p65 (green) to cell nuclei countersatained with DAPI (blue). Representative images from confocal microscopy of p65 immunofluorescence staining are shown. Scale bar represents 50 μm (G). For quantification of cells with nuclear localization of p65 at least 200 cells per treatment were counted (n = 4). For Western blots, representative gels are shown on top of the corresponding graphs, with β-actin used as a loading control. Bar graphs represent mean ± SEM. Statistical differences were analyzed by t-test. *p < 0.05 versus unstimulated control