Fig 2: Effects of ASM blockade on NLRP3 inflammasome formation in the primary cultures of ECs from EC-specific Smpd1 transgenic mice. Primary cultured carotid arterial ECs were treated with 7-Ket (15 µg/ml) for 24 h followed by treatment of ASM inhibitor Ami (20 µmol/l). A: Representative fluorescent confocal microscope images displaying the yellow dots or patches showing the colocalization of NLRP3 (green) with ASC (red). B: The summarized data show the colocalization coefficient of NLRP3 with ASC. C: Representative fluorescent confocal microscope images showing the colocalization of NLRP3 (green) with caspase-1 (red). D: The summarized data show the colocalization coefficient of NLRP3 with caspase-1. Data are expressed as means ± SEM, n = 6. *P < 0.05 is defined as significant. The scale bar represents 10 µm.

Fig 3: Acid sphigomyelinase is a target of penfluridol. A Raw 264.7 cells were collected to perform DARTS assay. B Raw 264.7 cells were used to conducted ASM activity assay. Cells were treated in presence or absence of TNFa (10 ng/mL) overnight, then lysed by freeze-thaw in liquid nitrogen for three cycles, followed by collecting supernatants after centrifuging for activity assay. C–J Flow cytometry to test effects of penfluridol on spleen naïve CD4+T cell differentiation into T cell subsets. C Differentiation from naïve CD4+T to TH1. D Quantification of C. E Differentiation from naïve CD4+T to TH2. F Quantification of E. G Differentiation from naïve CD4+T to TH17. H Quantification of G. I Differentiation from naïve CD4+T to Treg. J quantification of I. Experiments were performed for 3 biological replications and one-way analysis of variance and Bonferroni post hoc test were used to test statistical significance of the differences among groups. (NS = not significance, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). PF: penfluridol

Fig 4: Aggregation and activation of NOXs in MR signaling platforms in the primary cultures of ECs from EC-specific Smpd1 transgenic mice. A: Representative fluorescent confocal microscopic images showing the colocalization of MR marker, CTXB (green) with NOX subunit, gp47phox (red). B: Summarized data showing the colocalization coefficient of CTXB with gp47phox. C: Representative fluorescent confocal microscopic images showing the colocalization of LR marker, CTXB (green) with NOX subunit, gp91phox (red). D: Summarized data showing the colocalization coefficient of CTXB with gp91phox. E: NOX-mediated O2•- production as measured by ESR spectrometry. Data are expressed as means ± SEM, n = 5. *P < 0.05 is defined as significant. The scale bar represents 10 µm.

Fig 5: Atherosclerotic lesions in the carotid arteries in EC-specific Smpd1 transgenic mice during hypercholesterolemia. A: H&E staining showing the neointima and media on the mouse carotid arterial wall. AOI: the media area (black arrowheads) and the intima area (white arrowheads). B: Quantitative analysis of atherosclerotic lesions in PLCA represented by calculation of the ratio between arterial intima and media area. C: Trichrome staining showing the collagen deposition on the mouse carotid arterial wall. D: Quantification of the blue color of Trichrome staining. E: Representative microscopic images of tissue slide with immunohistochemical staining that shows a-SMA expression on the arterial wall. F: Summarized data showing the density of a-SMA stained with anti-a-SMA antibody. Data are expressed as means ± SEM, n = 5. *P < 0.05 is defined as significant. The scale bar represents 100 µm.