Fig. 2: A30L activates the NLRP3 inflammasome in human monocytes.

a Boxplot of IL1B expression levels in uninfected and MPXV-infected HeLa cells (from the GSE36854 cohort). b IL-1β and TNF concentrations in the supernatants of the indicated cells (human PBMCs and monocytes) stimulated with LPS (10 ng/mL for 4 h) followed by A30L (5 μg/mL for 18 h) were measured via ELISA. c IL-1β concentrations in the supernatants of PMA-differentiated THP-1 cells stimulated with LPS (10 ng/mL for 4 h) followed by A30L (5 μg/mL for 18 h), as measured by ELISA. d IL-1β concentrations in the supernatants of murine BMDMs and PMs stimulated with A30L (with the indicated concentration for 18 h), as measured by ELISA. e Western blot analysis of mature IL-1β and caspase-1 levels in culture supernatants (Sup.) and pro-IL-1β, pro-caspase-1, NLRP3, and ASC levels in cell lysates (Lys.) from human primary monocytes treated with A30L (5 μg/mL for 18 h) after LPS priming. f Western blot analysis of NLRP3 and mature IL-1β levels in human primary monocytes treated with A30L (5 μg/mL for 18 h) after NLRP3 knockdown and LPS priming. g IL-1β concentrations in the supernatants of monocytes stimulated with LPS (10 ng/mL for 4 h) followed by A30L (5 μg/mL for 18 h) after NLRP3 knockdown, as measured by ELISA. Representative images of immunofluorescence staining (scale bar = 20 μm; h) and quantitative analysis of the relative fluorescence intensities of DAPI and mtROS (i) in A30L-treated human primary monocytes (5 μg/mL for 4 h) after pretreatment with MitoTEMPO (100 μM for 2 h). The mtROS and nuclei were stained with MitoSOX Red (red) and DAPI (blue), respectively. IL-1β concentrations in the culture supernatants of cells incubated in the presence or absence of MitoTEMPO (j) or KCl (k). Statistical analysis was performed with an unpaired t-test with the Mann‒Whitney test or one-way ANOVA with Tukey’s multiple comparison test, and the results are presented as the means ± SDs. *p < 0.05; **p < 0.01; ***p < 0.001. ns not significant.