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. 2022 Oct 31;220(1):e20220837. doi: 10.1084/jem.20220837

Figure 5.

Figure 5.

Alphavirus infection and cytosolic dsRNA activate human NLRP1 in a p38 and ZAKα-dependent manner. (A–C) HEKNLRP1+ASC (A) or N/TERT-1C1C-EGFP (B and C) cells were infected with SFV at an MOI of 5 for 20 h in the presence of DMSO, 20 µM SB, 10 µM Dora, 1 μM MLN4, 1 μM PF, 200 nM ISRIB, 3 µM Jnk, 100 nM ZAKα inhibitor 6p (ZAKi), or 100 μM VX. Infected cells were stained with antibodies for dsRNA. Infection and speck assembly in infected cells were quantified by flow cytometry as described in Fig. 4. IL-1β from the supernatants of cells was quantified by HTRF (C). (D) N/TERT-1C1C-EGFP were treated with DMSO, 15 µM Aniso, or 30 μM Tal for the indicated times in the presence of DMSO, 10 µM Dora, or 100 nM ZAKi. Lysates were analyzed by immunoblot with antibodies for p38, P-p38, MKK3, P-MKK3, NLRP1, and Vinculin. (E) N/TERT-1C1C-EGFP cells and their polyclonal ZAKα or TAOK2 knockout derivatives were infected with SFV as in A, or stimulated with DMSO, Aniso, or Tal as in D for the indicated times. Infection and speck assembly in infected, C1C-EGFP–positive cells (SFV), or speck assembly in C1C-EGFP–positive cells (untreated and other triggers) were quantified as before. (F) Polyclonal ZAKα or TAOK2 knockout derivatives of N/TERT-1C1C-EGFP cells were analyzed by immunoblot with the indicated antibodies. (G) N/TERT-1C1C-EGFP cells were transfected with 1 µg/ml of the indicated nucleic acid species in presence of DMSO, 20 µM SB, 10 µM Dora, 100 nM ZAKi, or 100 μM VX for 20 h. Speck assembly and IL-1β release were quantified as in Fig. 1. (H) HEKNLRP1+ASC cells transiently expressing FLAG-tagged HRV14 protease 3C in the presence of 20 μM SB, 10 µM Dora, 1 μM MLN4, 1 μM PF, 200 nM ISRIB, 3 µM Jnk, or DMSO for 20 h were analyzed for FLAG expression and specks as described in Fig. 1. 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 or IL-1β release represents average values (with individual data points) from three independent experiments ± SEM. Immunoblots in D and F display data representative of three independent experiments. MW, molecular weight. Source data are available for this figure: SourceData F5.