Fig. 3.

Human variants phenocopy corresponding mouse variants. a Nlrp3^−/− iBMDMs with stably integrated human NLRP3 variants were primed with LPS (100 ng/ml) and doxycycline (1 μg/ml) for 11 h and stimulated with nigericin (10 μM) or ATP (5 mM) for 45 min. Supernatants were analyzed for IL-1β maturation (left) and LDH activity (right). b Cells were unprimed (doxycycline-treated) or primed (LPS, doxycycline) for 12 h and stimulated for 1 h, and cell supernatants were analyzed for mature IL-1β and caspase-1 p20 subunit. Cell lysates were analyzed for expression of pro-caspase-1, pro-IL-1β, ASC, and NLRP3 variant. c Supernatants from b were analyzed for TNF-α and IL-6 concentrations. d, e LPS-primed and nigericin-treated Nlrp3^−/− iBMDMs with human NLRP3 were analyzed for the formation of ASC specks. Nuclei are depicted in blue (DAPI), ASC in green, and actin in red; the bar represents 10 μm. To provide an estimate of ASC speck formation (in %) (e), four random 138 × 110 μm² frames were recorded for each condition, the number of ASC specks was divided by the number of nuclei within each frame. Representative of 3 (a–e) independent experiments is shown. The mean and the s.e.m. of 3 (a) or 2 (c) biological replicates and 4 random frames (e) are shown. 1–688 variant corresponds to MiniNLRP3