Extended Data Fig. 7: NEK7 is not required for activity of nigericin-sensitive NLRP3 variants.
a. Immunoblot analysis of NEK7, NLRP3 and β-actin in WT THP-1 cells or CRISPR-knockout of NEK7 or NEK7 and NLRP3 together. b. Release of IL-1β and IL-18 from wild-type (WT), NEK7–/–, NLRP3–/–NEK7–/– THP-1 cells left untreated, or primed with Pam3CSK4 (Pam3, 100 ng/mL) for 14 hours and then with nigericin (Nig., 10 μM) for 1 hour. c-d. Percentage of cells expressing GFP (c) and the release of IL-1β and IL-18 (d) in NLRP3–/–or NLRP3–/–NEK7–/– THP-1 cells reconstituted with GFP-tagged wild-type or G757R left untreated or primed with Pam3CSK4 (Pam3, 100 ng/mL) for 14 hours followed by 6 hours of doxycycline (Dox, 100 ng/mL) treatment. e-h. Percentage of cells expressing GFP and the release of IL-1β and IL-18 in NLRP3–/–NEK7–/– THP-1 cells reconstituted with GFP-tagged wild-type (WT), Y861H (e, f) or R920Q (g, h), untreated or primed with Pam3CSK4 (Pam3, 100 ng/mL) for 14 hours followed by 6 hours of doxycycline (Dox, 100 ng/mL) treatment and increasing concentrations of nigericin (Nig., 0.5 μM, 1 μM, 5 μM,10 μM) for 1 hour. Data was representative of 2 independent repeats (a), or pooled from 3 independent repeats (b-h). Each dot represents a single repeat (b-h, mean and s.e.m. in b-h). Two-tailed t-test in c, e, g, one-way ANOVA with Dunnett’s multiple-comparisons test in b, d, f, h.
