Figure S1.
Reporter cell lines recapitulate NLRP1 inflammasome assembly. (A) Lysates of HEK 293T (HEK), HEKNLRP1+ASC, and HEKNLRP3+ASC cells were analyzed by immunoblot with the indicated antibodies to confirm expression of NLRP1-HA, NLRP3-HA, and ASC-EGFP. (B and F) HEKNLRP1+ASC or N/TERT-1C1C-EGFP cells were seeded on cover slips and stimulated with DMSO or 30 µM Tal for 20 h as in (Fig. 1, B and H). Fixed cells were stained for DNA and representative images were recorded by wide-field fluorescence microscopy. Scale bars represent 50 μm. (C and K) HEKNLRP1+ASC, HEKNLRP3+ASC, or N/TERT-1C1C-EGFP cells were treated with DMSO or 10 mM sodium azide and 50 mM 2-DG for 6 h. ASC-EGFP or C1C-EGFP speck formation was quantified by flow cytometry as described in Fig. 1, A and F. N/TERT-1C1C-EGFP cells were stimulated in the presence of 100 µM VX for flow cytometry experiments. IL-1β from the supernatants of cells stimulated in the absence or presence of 100 μM VX was quantified by HTRF. (D) HEKNLRP3+ASC cells were treated with 200 ng/ml LPS for 3 h and 10 µM Nig for 1 h, followed by quantification of ASC-EGFP specks by flow cytometry as in C. (E) Cell lysates of N/TERT-1 and N/TERT-1C1C-EGFP cells were analyzed by immunoblot with the indicated antibodies to confirm expression of NLRP1 and C1C-EGFP. (G–J) N/TERT-1C1C-EGFP cells and their monoclonal NLRP1 knockout derivatives were stimulated with 30 µM Tal (G and I), UV for 3 min (H and I), or transfected with 1 µg/ml of the indicated nucleic acid species (J) for 20 h as in Fig. 1, H, J, and K, where indicated in the presence of DMSO, 100 μM VX, or 50 μM Z-VAD. Cell death was quantified by detection of LDH release into the supernatant (G–J), or by uptake of non-cell permeable DNA dye DRAQ7 over 20 h (I). For LDH detection, the same supernatants as for IL-1β detection in Fig. 1, H, J, and K, were used. Data from all experiments quantifying specks, LDH release, or IL-1β release represents average values (with individual data points) from three independent experiments ± SEM. Immunoblots in A and E, microscopy images in B and F and quantifications of DRAQ7 uptake over time in I display experiment representatives of two or three independent experiments. MW, molecular weight. Source data are available for this figure: SourceData FS1.

Reporter cell lines recapitulate NLRP1 inflammasome assembly. (A) Lysates of HEK 293T (HEK), HEKNLRP1+ASC, and HEKNLRP3+ASC cells were analyzed by immunoblot with the indicated antibodies to confirm expression of NLRP1-HA, NLRP3-HA, and ASC-EGFP. (B and F) HEKNLRP1+ASC or N/TERT-1C1C-EGFP cells were seeded on cover slips and stimulated with DMSO or 30 µM Tal for 20 h as in (Fig. 1, B and H). Fixed cells were stained for DNA and representative images were recorded by wide-field fluorescence microscopy. Scale bars represent 50 μm. (C and K) HEKNLRP1+ASC, HEKNLRP3+ASC, or N/TERT-1C1C-EGFP cells were treated with DMSO or 10 mM sodium azide and 50 mM 2-DG for 6 h. ASC-EGFP or C1C-EGFP speck formation was quantified by flow cytometry as described in Fig. 1, A and F. N/TERT-1C1C-EGFP cells were stimulated in the presence of 100 µM VX for flow cytometry experiments. IL-1β from the supernatants of cells stimulated in the absence or presence of 100 μM VX was quantified by HTRF. (D) HEKNLRP3+ASC cells were treated with 200 ng/ml LPS for 3 h and 10 µM Nig for 1 h, followed by quantification of ASC-EGFP specks by flow cytometry as in C. (E) Cell lysates of N/TERT-1 and N/TERT-1C1C-EGFP cells were analyzed by immunoblot with the indicated antibodies to confirm expression of NLRP1 and C1C-EGFP. (G–J) N/TERT-1C1C-EGFP cells and their monoclonal NLRP1 knockout derivatives were stimulated with 30 µM Tal (G and I), UV for 3 min (H and I), or transfected with 1 µg/ml of the indicated nucleic acid species (J) for 20 h as in Fig. 1, H, J, and K, where indicated in the presence of DMSO, 100 μM VX, or 50 μM Z-VAD. Cell death was quantified by detection of LDH release into the supernatant (G–J), or by uptake of non-cell permeable DNA dye DRAQ7 over 20 h (I). For LDH detection, the same supernatants as for IL-1β detection in Fig. 1, H, J, and K, were used. Data from all experiments quantifying specks, LDH release, or IL-1β release represents average values (with individual data points) from three independent experiments ± SEM. Immunoblots in A and E, microscopy images in B and F and quantifications of DRAQ7 uptake over time in I display experiment representatives of two or three independent experiments. MW, molecular weight. Source data are available for this figure: SourceData FS1.

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