Extended Data Fig. 3: NLRP3 reconstitution in THP-1 cells. | Nature Immunology

Extended Data Fig. 3: NLRP3 reconstitution in THP-1 cells.

From: Mechanisms of NLRP3 activation and inhibition elucidated by functional analysis of disease-associated variants

Extended Data Fig. 3

a. Immunoblot analysis of NLRP3 and tubulin in WT THP-1 cells or NLRP3 CRISPR-knockout using guide 1 or 2 (sg1 or 2). b. Release of IL-1β, IL-18 and IL-6 from NLRP3–/–THP-1 cells left untreated, or primed with Pam3CSK4 (Pam3, 100 ng/mL) for 14 hours and then with nigericin (Nig., 10 μM) for 3 hours or primed with Pam3CSK4 (Pam3, 100 ng/mL) and IFN-γ (1 μg/mL) for 24 hours and then treated with TcdB (200 ng/mL) for 14 hours. c. Release of IL-1β and IL-18 in NLRP3–/– or NLRP3–/– THP-1 cells reconstituted with GFP-tagged wild-type (WT) NLRP3 left untreated or treated with Pam3CSK4 (Pam3, 100 ng/mL) for 14 hours followed by doxycycline (Dox, 100 ng/mL) for 6 hours with or without MCC950 (10 μM), and then nigericin (Nig., 10 μM) for 1 hour. d. Percentage of cells expressing GFP and the release of IL-1β and IL-18 in NLRP3–/– THP-1 cells reconstituted with GFP-tagged wild-type (WT), R262P, R262L or R262W NLRP3 after treatment with Pam3CSK4 (Pam3, 100 ng/mL) for 14 hours and doxycycline (Dox, 100 ng/mL) for 6 hours. Data are representative of two independent repeats (a) or collective of three independent repeats (b-d, mean and s.e.m. in b-d). One-way ANOVA with Dunnett’s multiple-comparisons test in b, d.

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