Figure S2.
Human NLRP1 is activated by the ribotoxic stress response. (A) HEKNLRP1+ASC cells were stimulated with 30 µM Tal, UV for 3 min, 20/2 µM Doxo, or 100/25 µM Eto and harvested after 20 h. Cells were stained for phospho-γH2A.X and analyzed for DNA damage markers (left) or ASC-EGFP specking (right) by flow cytometry as in Fig. 1. (B) HEKNLRP1+ASC cells were stimulated with UV for 3 min and subsequently cultivated in the presence of 10/1 µM KU-60019 (KU), or 1/0.1 µM Berzosertib (Berz) for 20 h, followed by quantification of ASC specks as in A. (C and D) HEKNLRP1+ASC, HEKNLRP3+ASC (C), or N/TERT-1C1C-EGFP (D) cells were treated with 1.5/0.3/0.06 mM H2O2 for 6 h, where indicated in the presence of 20 μM SB or 100 μM VX. Specks and secreted IL-1β were quantified by flow cytometry and HTRF, respectively. (E and F) HEKNLRP1+ASC, HEKNLRP3+ASC (E), or N/TERT-1C1C-EGFP (F) cells were treated with DMSO or 15/1.5/0.15 µM Aniso for 20 h, where indicated in the presence of 20 µM SB. Specks were quantified as in A. (G and H) HEKNLRP1+ASC (G) or N/TERT-1C1C-EGFP (H) cells were seeded on cover slips and stimulated with DMSO or 15 µM Aniso for 6 h as in (Fig. 2, G and I). Fixed cells were stained for DNA and representative images were recorded by wide-field fluorescence microscopy. Scale bars represent 50 μm. (I–K) HEKNLRP1+ASC, HEKNLRP3+ASC (I), or N/TERT-1C1C-EGFP (J and K) cells were treated with DMSO, 15/1.5/0.15 µM Aniso (K), 1,000/200/40 µM CHX (I–K), or 40/4/0.8 µg/ml Blasti (I–K) for 20 h where indicated in the presence of 20 μM SB or 100 μM VX. Specks and secreted IL-1β were quantified by flow cytometry and HTRF. ASC speck assembly after Aniso treatment shown in F was done in the same experiment. (L and M) N/TERT-1C1C-EGFP cells and their monoclonal ASC or NLRP1 knockout derivatives were stimulated with DMSO, 15 µM Aniso, or 2 µM Lacti for 20 h as in Fig. 2 K, where indicated in the presence of DMSO, 100 μM VX, 50 μM Z-VAD, or 10 μM Dora. Cell death was quantified by detection of LDH release (L) or uptake of non-cell permeable DNA dye DRAQ7 over 20 h (M). For LDH detection, the same supernatants as for IL-1β detection in Fig. 2 K were used. (N) Lysates of N/TERT-1C1C-EGFP or its polyclonal ASC, p38α, or p38β knockout derivatives were analyzed by immunoblot with the indicated antibodies. N/TERT-1C1C-EGFP cells were stimulated in the presence of 100 µM VX for all flow cytometry experiments. Data from all experiments quantifying specks, LDH release, or IL-1β release represents average values (with individual data points) from one or three independent experiments ± SEM. Microscopy images in G and H, quantifications of DRAQ7 uptake over time in M and immunoblots in N display experiment representatives of two or three independent experiments. MW, molecular weight. Source data are available for this figure: SourceData FS2.

Human NLRP1 is activated by the ribotoxic stress response. (A) HEKNLRP1+ASC cells were stimulated with 30 µM Tal, UV for 3 min, 20/2 µM Doxo, or 100/25 µM Eto and harvested after 20 h. Cells were stained for phospho-γH2A.X and analyzed for DNA damage markers (left) or ASC-EGFP specking (right) by flow cytometry as in Fig. 1. (B) HEKNLRP1+ASC cells were stimulated with UV for 3 min and subsequently cultivated in the presence of 10/1 µM KU-60019 (KU), or 1/0.1 µM Berzosertib (Berz) for 20 h, followed by quantification of ASC specks as in A. (C and D) HEKNLRP1+ASC, HEKNLRP3+ASC (C), or N/TERT-1C1C-EGFP (D) cells were treated with 1.5/0.3/0.06 mM H2O2 for 6 h, where indicated in the presence of 20 μM SB or 100 μM VX. Specks and secreted IL-1β were quantified by flow cytometry and HTRF, respectively. (E and F) HEKNLRP1+ASC, HEKNLRP3+ASC (E), or N/TERT-1C1C-EGFP (F) cells were treated with DMSO or 15/1.5/0.15 µM Aniso for 20 h, where indicated in the presence of 20 µM SB. Specks were quantified as in A. (G and H) HEKNLRP1+ASC (G) or N/TERT-1C1C-EGFP (H) cells were seeded on cover slips and stimulated with DMSO or 15 µM Aniso for 6 h as in (Fig. 2, G and I). Fixed cells were stained for DNA and representative images were recorded by wide-field fluorescence microscopy. Scale bars represent 50 μm. (I–K) HEKNLRP1+ASC, HEKNLRP3+ASC (I), or N/TERT-1C1C-EGFP (J and K) cells were treated with DMSO, 15/1.5/0.15 µM Aniso (K), 1,000/200/40 µM CHX (I–K), or 40/4/0.8 µg/ml Blasti (I–K) for 20 h where indicated in the presence of 20 μM SB or 100 μM VX. Specks and secreted IL-1β were quantified by flow cytometry and HTRF. ASC speck assembly after Aniso treatment shown in F was done in the same experiment. (L and M) N/TERT-1C1C-EGFP cells and their monoclonal ASC or NLRP1 knockout derivatives were stimulated with DMSO, 15 µM Aniso, or 2 µM Lacti for 20 h as in Fig. 2 K, where indicated in the presence of DMSO, 100 μM VX, 50 μM Z-VAD, or 10 μM Dora. Cell death was quantified by detection of LDH release (L) or uptake of non-cell permeable DNA dye DRAQ7 over 20 h (M). For LDH detection, the same supernatants as for IL-1β detection in Fig. 2 K were used. (N) Lysates of N/TERT-1C1C-EGFP or its polyclonal ASC, p38α, or p38β knockout derivatives were analyzed by immunoblot with the indicated antibodies. N/TERT-1C1C-EGFP cells were stimulated in the presence of 100 µM VX for all flow cytometry experiments. Data from all experiments quantifying specks, LDH release, or IL-1β release represents average values (with individual data points) from one or three independent experiments ± SEM. Microscopy images in G and H, quantifications of DRAQ7 uptake over time in M and immunoblots in N display experiment representatives of two or three independent experiments. MW, molecular weight. Source data are available for this figure: SourceData FS2.

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