Fig. 6.
MAVS is required for Pnpt1-regulated NLRP3 inflammasome activation. A Double-stranded RNA was measured in untreated BMDMs from WT and Pnpt1m−/− mice by immunofluorescence (J2 antibody, green) and DAPI (blue), scale bar: 5 µm (B) The ratio of total J2-positive cells/DAPI-positive cells was quantified by ImageJ. HEK-293T cells were transfected with scramble (control), Pnpt1 or MAVS siRNA for 48 h, and gene expression was measured by real-time PCR. C ISG-15 and (D) IFN-β mRNA levels in cell lysates. E BMDMs were treated with scrambled or MAVS siRNA for 48 h and stimulated with LPS (100 ng/mL) for 3 h, followed by nigericin (Nig, 2 µM) stimulation for 1 h. F THP-1 macrophages were treated with scrambled, Pnpt1 or MAVS siRNA for 48 h and stimulated with LPS (100 ng/mL) for 3 h, followed by 10 µg/mL poly(I:C) transfection (iC) for 12 h. The amount of IL-1β in the medium was measured. G The amount of ISG mRNA in cell lysates after stimulation with LPS plus poly (I:C) in cells treated with scrambled or MAVS siRNA was measured (n = 4 experiments, control: con; knockdown: KD). Statistical analyses in (B) were performed using an unpaired t-test. Statistical analyses in (C–G) were performed using a 2-way ANOVA and Bonferroni’s post hoc test. Bars represent the mean ± SEM. ***p < 0.001, ****p < 0.001