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. 2024 Apr 25;20(4):e1012167. doi: 10.1371/journal.ppat.1012167

Fig 1. DENV NS1 can activate the inflammasome.

Fig 1

(A) BMDMs were primed with PAM3CSK4 (1μg/mL) for 17 h and then treated with DENV2 NS1 at indicated concentrations, treated with 5μM nigericin, or left untreated (PAM only). IL-1β levels in the supernatant after 2h (nigericin) or 24h (NS1 and PAM only) were measured by ELISA. Statistical significance was determined by one-way ANOVA with Dunn’s multiple compa rison correction. (B) Representative Western blots of BMDM cell lysates after priming with PAM3CSK4 (1μg/mL) for 17h and treatment with 10ug/mL DENV2 NS1 (DENV NS1) or PAM3CSK4 treatment for 24h without NS1 treatment (PAM only). (C) WT and Casp1/11-/- BMDMs were primed with PAM3CSK4 (1μg/mL) for 17h and then treated with DENV2 NS1 at the indicated concentrations, nigericin (5μM), or medium (PAM only). IL-1β levels in the supernatant after 2h (nigericin) or 24h (NS1 and PAM only) were measured by ELISA. Statistical significance was determined using two-way ANOVA with Holm-Sidak’s multiple comparisons test. (D) BMDMs were primed with PAM3CSK4 (1μg/mL) for 17h and then pre-treated with Ac-YVAD-cmk at the indicated concentrations before addition of DENV2 NS1 (10μg/mL), nigericin (5μM), or medium (Inhibitor only). IL-1β levels in supernatant 2h (nigericin) or 24h (NS1 and PAM only) were measured by ELISA. (E) WT or Casp-1-/- THP-1 human monocytes were differentiated into macrophages in 10ng/mL PMA and primed with medium or LPS for 4h. Primed macrophages were treated with DENV NS1 (10μg/mL) or left untreated (LPS only). Eighteen hours later, supernatants were collected. Cells were stimulated with 5μM nigericin for 2h as a positive control. Bioactive IL-1 in supernatants was measured using HEK-Blue IL-1R reporter cells. Statistical significance was determined using two-way ANOVA with Holm-Sidak’s multiple comparisons test (F) Representative Western blots of THP-1 macrophage cell lysates after priming with PAM3CSK4 (100ng/mL) for 17h and treatment with DENV2 NS1 at indicated concentrations (μg/mL), treatment with 5μM nigericin, or no treatment for 24h. The data are shown as the mean ± standard deviation (SD) of 3 independent experiments (A, C-D) or 4–6 independent experiments (E) or a representative image taken from 2 biological replicates (B, F). *p<0.05, **p<0.01, *** p< 0.001, ****p<0.0001.