Figure 2. DENV NS1-induced inflammasome activation is NLPR3-independent.

(A) WT and Nlrp3 ^−/− BMDMs were primed with PAM₃CSK₄ (1μg/mL) for 17h and then treated with DENV2 NS1 at indicated concentrations, nigericin (5μM), or medium (PAM only). IL-1β levels in supernatant 2h (nigericin) or 24h (NS1 and PAM only) were measured by ELISA. *p<0.05, **p<0.01. Statistical significance was determined using two-way ANOVA followed by multiple t-tests with Holm-Sidak correction. (B) Representative Western blots of cell lysates from WT and Nlrp3^−/− BMDMs after priming with PAM₃CSK₄ (1μg/mL) for 17h and treatment with DENV2 NS1 (10 or 5 μg/mL), treatment with nigericin (5μM), or no treatment for 24h. (C) BMDMs were primed with PAM₃CSK₄ (1μg/mL) for 17h and then pre-treated with MCC950 at the indicated concentrations before addition of DENV2 NS1 (10μg/mL), nigericin (5μM), or medium (Inhibitor only). IL-1β levels in the supernatant after 2h (Nigericin) or 24h (NS1 and PAM only) were measured by ELISA. (D) Representative Western blots of cell lysates from BMDMs nucleofected with Cas9-gRNA ribonuclear protein complexes to knock out the indicated genes. Two gRNAs per gene were used per nucleofection. NTG = non-targeting guide. (E) Knockout BMDMs from (D) were primed with PAM₃CSK₄ (1μg/mL) for 17h and treated with DENV2 NS1 (10μg/mL) or left untreated for 48h. *p<0.05. Statistical significance was determined using two-way ANOVA followed by multiple t-tests with Holm-Sidak correction. The data are shown as the mean ± SD of 3 biological replicates (A,C), a representative image taken from 2 biological replicates (B,D), or data pooled from 5 independent experiments with 3 biological replicates per guide (E).