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. 2024 Feb 28;10(9):eadj6289. doi: 10.1126/sciadv.adj6289

Fig. 3. SGT1 and HSP90β facilitate inflammasome speck formation in NLRP3 CAPS model.

Fig. 3.

(A) ASC oligomerization in U937 NLRP3 R260W control cells (sgCTRL) or lacking SGT1 (sgSUGT1) or HSP90β (sgHSP90ab1). Cells are treated with doxycycline overnight or left untreated. Protein lysates are cross-linked and analyzed by Western blot. (B) SGT1 (sgSUGT1)– or HSP90β (sgHSP90ab1)–deficient U937 NLRP3 R260W clones or control populations (sgCTRL) were treated with doxycycline and z-vad-fmk for 24 hours and intracellularly stained using anti-ASC antibodies. Hoechst was added to stain nuclei. Cells are measured using imaging flow cytometry. Representative images with ASC specks in sgCTRL or ASC diffuse pattern in sgSUGT1 cells are shown. Bottom shows the quantification of ASC speck formation in the tested conditions. (C and D) ASC oligomerization in U937 NLRP3 R260W control cells (sgCTRL) or lacking SGT1 (sgSUGT1) or HSP90β (sgHSP90ab1). Cells are treated with nigericin (Nig.), doxycycline, or both. Cells were either lysed for protein cross-linking and analyzed by Western blot (C) or fluorescently stained and analyzed by imaging flow cytometry for ASC speck formation (D). (E) Inflammasome activation in U937 NLRP3 R260W control cells (sgCTRL) or lacking SGT1 (sgSUGT1) or HSP90β (sgHSP90ab1). Cells are treated either with PMA and doxycycline or with PMA and nigericin. The release of cleaved IL-1β in the supernatant is quantified by ELISA. Data are obtained from three independent experiments, and represented as means ± SD, and tested for statistical significance using the nonparametric Mann-Whitney U test (P ≤ 0.05 is considered significant and indicated with *).