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. 2021 Dec 5;75(1):196–212. doi: 10.1002/hep.32114

FIGURE 2.

FIGURE 2

Inoculation of HEV activates NLRP3 inflammasome in human primary macrophages and macrophage cell lines. (A) Schematic illustration of generating primary macrophages from PBMC‐derived monocytes. Primary macrophages and HL60 macrophages were inoculated with HEV for 24 hours or treated with LPS (400 ng/mL) for 6 hours, followed by ATP (5 mM) for 40 minutes. IL‐1β mRNA (B,D) and protein (C,E) levels were quantified by qRT‐PCR (n = 4‐6) and ELISA (n = 4‐6), respectively. THP‐1 macrophages were inoculated with HEV for 12, 24, or 48 hours or treated with LPS (400 ng/mL) for 6 hours, followed by ATP (5 mM) for 40 minutes. IL‐1β mRNA (F) and protein (G) levels were quantified by qRT‐PCR (n = 8) and ELISA (n = 4), respectively. (H) Mature IL‐1β and cleaved Casp‐1 in supernatant or pro‐IL‐1β, pro‐Casp‐1, and NLRP3 in lysates were determined by western blotting. (I) THP‐1 macrophages were inoculated with HEV for 24 hours or treated with LPS (400 ng/mL) for 6 hours, followed by ATP (5 mM) for 40 minutes. Subcellular localizations of NLRP3 (green) and the nucleus marker, DAPI (blue), were examined under confocal microscopy. (J) THP‐1 macrophages were pretreated with 50 µM of Casp‐1 inhibitor (VX‐765) or 10 µM of NLRP3 inhibitor (MCC950) for 2 hours and then were treated with LPS (400 ng/mL) or LPS (400 ng/mL) plus 50 µM of Casp‐1 inhibitor (VX‐765) or 10 µM of NLRP3 inhibitor (MCC950) for 6 hours, followed by ATP (5 mM) for 40 minutes. IL‐1β level in supernatant was quantified by ELISA (n = 4). (K) THP‐1 macrophages were pretreated with 50 µM of Casp‐1 inhibitor (VX‐765) or 10 µM of NLRP3 inhibitor (MCC950) for 2 hours and then were incubated with HEV plus 50 µM of Casp‐1 inhibitor (VX‐765) or 10 µM of NLRP3 inhibitor (MCC950) for 24 hours. IL‐1β level in supernatant was quantified by ELISA (n = 4). NLRP3 inflammasome is marked by an arrow. Data were normalized to the control (CTR; set as 1). Data are means ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Abbreviations: NS, not significant (Mann‐Whitney U test); Super, supernatant