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. Author manuscript; available in PMC: 2022 Nov 2.
Published in final edited form as: Cell Rep. 2022 Oct 11;41(2):111472. doi: 10.1016/j.celrep.2022.111472

Figure 4. Pyrin inflammasome activation proceeds differently following TcdA/B and steroid catabolite addition.

Figure 4.

(A) Monocytes from HDs (n = 2–12) were treated with colchicine or nocodazole and 30 min later with the indicated stimuli. Propidium iodide incorporation was monitored every 5 min for 3 h.

(B) The area under the cure (AUC) is shown. For each HD, the AUC values in the presence of inhibitors were normalized to the AUC value obtained in the absence of inhibitor.

(C) Monocytes from HDs (n = 8–14) were treated with colchicine and 30 min later with the indicated stimuli. IL-1β concentration in the supernatant was quantified at 3 h post-addition.

(D) RhoA activity was determined by G-LISA at different time post-treatment in the lysate of U937 cells. The activity of the different treatments at the indicated time is presented in Figure S4B.

(E) Doxycycline-induced, PMA-differentiated U937 macrophages expressing MEFV Full-length (FL), deleted of the pyrin (ΔPYD), of the phosphorylated linker (ΔPLD), of the BBox (ΔBbox), of the coiled-coil (ΔCC), or the B30.2 (ΔB30.2) domains were treated with LPS for 3 h and then with the indicated stimuli. IL-1β concentration in the supernatant was quantified at 3 h post-addition.

(F) Doxycycline-induced, U937 monocytes expressing WT or ΔB30.2 MEFV were treated with the indicated stimuli for 90 min. Pyrin S242 phosphorylation was assessed by western blot analysis following immunoprecipitation. “*” indicates a cleaved form of pyrin.

(A–E) Mean and SEM are shown. (B and C) Each dot represents the value for one HD. (D and E) One experiment with technical triplicates representative of two (D) to three (E) independent experiments is shown. (B–C) Wilcoxon matched-pairs signed rank tests were performed to compare values with or without colchicine/nocodazole. Two-tailed p values: (B) **p = 0.0078, ***p < 0.001; (C) ***p < 0.001, **p = 0.002; (D) ordinary one-way ANOVA with Holm-Sidak’s correction for multiple tests was performed. *p = 0.015, ***p < 0.001. (E) Ordinary one way ANOVA with Sidak’s correction for multiple tests was performed. *p = 0.011, ***p < 0.001.