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

Fig. 2. NLRP3 function is HSP90β and SGT1 selective.

Fig. 2.

(A and Β) Inflammasome activation in U937 NLRP3 R260W control cells or depleted for SGT1 (sgSUGT1) or reconstituted with SGT1b. Cells are treated with PMA and doxycycline overnight. (A) Protein expression and release of inflammasome activation markers are measured by Western blot. (B) Release of cleaved IL-1β in the supernatant is quantified by ELISA. (C and D) Inflammasome activation in U937 NLRP3 R260W control cells or depleted for HSP90β (sgHSP90ab1) or reconstituted with HSP90ab1. Cells are treated with PMA and doxycycline overnight. (C) Protein expression and release of inflammasome activation markers are measured by Western blot. (D) Release of cleaved IL-1β in the supernatant is quantified by ELISA. (E and F) Inflammasome activation in U937 NLRP3 R260W control cells (sgCTRL) or lacking HSP90α (sgHSP90aa1). Cells are treated with PMA and doxycycline overnight. (E) Protein expression and release of inflammasome activation markers are measured by Western blot. (F) Release of cleaved IL-1β in the supernatant of doxycycline-treated cells is quantified by ELISA. (G) Inflammasome activation in U937 MEFV M694V control cells or depleted for SGT1 (sgSUGT1) or HSP90β (sgHSP90ab1). Cells are treated with PMA and doxycycline overnight. Protein expression and release of inflammasome activation markers are measured by Western blot. (H) Cell death analysis of U937 MEFV M694V control cells or depleted for SGT1 (sgSUGT1) or HSP90β (sgHSP90ab1). Cells are treated with doxycycline for 24 hours, and PI-positive cells are measured by flow cytometry. Western blots are representative of three or four independent experiments. ELISA data are obtained from three or four independent experiments, 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 *; ns, not significant).