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. 2024 Dec 17;7:0551. doi: 10.34133/research.0551

Fig. 7.

Fig. 7.

HSP90α inhibition caused by polydatin posed a challenge to the stability of the NLRP3 CAPS-associated mutants. (A to H) NLRP3lyz−/− BMDMs were recombined with mouse NLRP3 L351P mutant (L351P-BMDM). (A) L351P-BMDMs were treated with indicated concentrations of polydatin followed by 100 ng/ml LPS for 3 h. IL-1β in the supernatant was determined by ELISA. (B) CASP1 activation was determined by FAM-FLICA staining. (C) Cleaved IL-1β (p17), activated CASP1 (p20) in culture supernatants (SN), and pro-IL-1β, IL-1β p17, pro-CASP1, and CASP1 p20 in lysates of NLRP3 L351P BMDMs were analyzed by immunoblot. (D) HEK293T cells were cotransfected with human NLRP3 (L353P), ASC, NEK7, and pro-CASP1 to reconstruct the human inflammasome system. CASP1 (p20) in culture supernatants and CASP1 (p20), pro-CASP1, and NLRP3 (L353P) in lysates were analyzed by immunoblot. (E) Western blot analysis of NLRP3 (L351P), ASC, NEK7, and pro-CASP1. (F) L351P-BMDMs were treated with indicated concentrations of polydatin or 10 μM GA for 1 h, followed by 100 ng/ml LPS for 3 h. The cells were then treated with 40 μg/ml digitonin on ice to permeabilize the plasma membrane. The NLRP3 (L351P) monomers and double-ring cage-NLRP3 oligomers were separated and examined by immunoblot. (G) L351P-BMDMs were treated with 20 μM MG132 and 30 μM CQ for 2 h, and then 10 μM polydatin or 10 μM GA treatment for 1 h followed by 100 ng/ml LPS for 3 h. The NLRP3 (L351P) monomers and double-ring cage-NLRP3 oligomers were separated and examined by immunoblot. (H) L351P-BMDMs were stimulated by 100 ng/ml LPS for 3 h. The cells were treated with 40 μg/ml digitonin on ice to permeabilize the plasma membrane. The pellets were treated with 10 μM polydatin or 10 μM GA for 1 h, and the NLRP3 (L351P) expression was analyzed by immunoblot. (I) An endogenous IP with HSP90α antibody was performed in LPS-primed BMDMs treated with or without CQ and MG132 in the presence of polydatin and GA. Western blot analysis of HSP90α and NLRP3 (L351P). Data are presented as mean ± SEM of 3 independent experiments. *P < 0.05, **P < 0.01 versus LPS.