Fig. 2

S protein promotes the activation of the NLRP3 inflammasome in HUVEC. Human umbilical vein endothelial cells (HUVEC) were treated with viral S protein (35 nM), IL-1β (2.5 ng/mL) or the S1 fragment of S protein (35 nM) for 18–24 h. A The formation of toroidal specks corresponding to activated NLRP3 inflammasome were visualized by indirect immunofluorescence against ASC (red) using a confocal microscope. Nuclei were counter-stained with DAPI (blue). Scale bar represents 50 μm B NLRP3 inflammasome activation was quantified by manual blind scoring of 27 radial distributed fields per sample as the number of ASC speck-positive cells. White arrows indicate speck positive cells. Scale bar represents 50 μm. In addition, the cellular protein levels of C active cleaved caspase-1 (casp-1) (n = 6) and D mature IL-1β (n = 4). E IL-1β released to the cell supernatants was quantified by ELISA (n = 5–8). F Gasdermin D (GSDMD) and cleaved GSDMD-NT (n = 8) were determined by Western blot in total cell lysates from HUVEC treated with 7, 35 and 70 nM S protein or 2,5 ng/mL IL-1β. 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 control with no S protein