Figure S2.

NLRP3 expression, priming, and/or activation triggers pyroptosis in reconstituted U937 depending on the NLRP3 variants. (A–F) NLRP3-deficient U937 cells reconstituted with doxycycline-inducible NLRP3 variants were treated with doxycycline (1 μg/ml, 3 h), LPS (40 ng/ml, 2 h), and nigericin (15 μg/ml) before cell death was monitored by PI incorporation over time quantified by time-lapse high content microscopy. (A) Unsupervised clustering of the variants according to RC from the glmm applied on AUC for each variant as compared with the WT. Positive RC denotes increased cell death in the considered variant as compared with the WT, and conversely. RC corresponding to increases (magenta) or decreases (cyan) in cell death are color-coded. Based on the results, variants are classified into five functional groups: constitutive active variants (group#5, red), variants active upon either priming or activation signal (group#4, green), variants active upon priming signal (group#3, yellow), variants active upon activation signal (group#2, blue), and mutants active upon priming and activation signals (no gain-of-function, group#1, black). Results of two to eight independent experiments done in duplicates. (B) Group#5. (C) Group#4. (D) Group#3. (E) Group#2. (F) Group#1. Means of duplicates and 1 SD are represented. One experiment done in duplicates representative of two to eight independent experiments is shown. Statistical analysis including all independent experiments are represented in Fig. 3. (G) NLRP3-deficient U937 cells reconstituted with doxycycline-inducible group#5 NLRP3 variants were treated with doxycycline (1 μg/ml) before cell death was monitored by PI incorporation over time quantified by time-lapse high content microscopy. One experiment done in duplicates representative of two independent experiments is shown. Statistical analysis including all independent experiments is represented in Fig. 2. (H–J) U937 cells transduced with vectors encoding indicated doxycycline-inducible NLRP3 variants or empty vector (empty) and not transduced controls (−) were treated with doxycycline (1 μg/ml, 4 h) or LPS (40 ng/ml, 3 h) and NLRP3 protein levels were analyzed by WB (H). Cells were treated with doxycycline (1 μg/ml, 3 h), LPS (40 ng/ml, 2 h), and/or nigericin (15 μg/ml) before monitoring PI incorporation (I). Means of duplicates and 1 SD are represented. One experiment done in duplicates representative of two to three independent experiments is shown. LDH release was assessed 2 h after nigericin treatment (J). Means of two independent experiments done in duplicates and 1 SD are represented. Source data are available for this figure: SourceData FS2.