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

Fig. 2.

Fig. 2.

Polydatin is a potential natural product that inhibited the formation of cage-NLRP3 and disc-NLRP3. (A) Schematic representation of screening natural products targeting cage-NLRP3 using flow cytometry. (B) HEK293T cells cotransfected with BIFC-nYFP-NLRP3 and BIFC-cYFP-NLRP3 were treated with different natural products, and the BIFC+ cells were determined by flow cytometry. The chemical structure of polydatin was shown. (C) HEK293T cells cotransfected with BIFC-nYFP-NLRP3 and BIFC-cYFP-NLRP3 were treated with indicated concentrations of polydatin, and the BIFC+ cells were determined by flow cytometry. (D) BMDMs were stimulated with 100 ng/ml LPS for 3 h, followed by indicated concentrations of polydatin treatment for 1 h. Oligomerization of NLRP3 in the Nonidet P40-insoluble pellet and NLRP3 in cell lysates were examined using Western blotting. (E) GFP-tagged vector. (F) Cage-NLRP3, (G) NLRP3-NEK7, and (H) NLRP3-NEK7-ASC complexes were reproduced in HEK293T cells. The effect of polydatin on 3 stages of NLRP3 complexes was observed by confocal microscopy. (I and J) LPS-primed BMDMs were treated with polydatin for 1 h, followed by ATP stimulation for 30 min. (I) ASC in cell lysates and oligomerization of ASC in the Nonidet P40-insoluble pellet were examined via Western blotting. (J) Oligomerization of NLRP3 was analyzed by blue native PAGE. (K) BMDMs were exposed to 100 ng/ml LPS for 3 h, followed by treatment with specified concentrations of polydatin for 1 h, and subsequently stimulated with 5 mM ATP for another 1 h. Western blotting analysis of NLRP3, ASC, NEK7, and pro-CASP1. (L) BMDMs were treated with 20 μM MG132 or 30 μM CQ for 2 h, and then 100 ng/ml LPS for 3 h followed by 10 μM polydatin treatment for 1 h. The expression of NLRP3 was determined by Western blot. (M and N) BMDMs were pretreated with CQ and MG132, followed by LPS and ATP stimulation, and an endogenous immunoprecipitation (IP) was performed using (M) ASC antibody or (N) NEK7 antibody, with or without polydatin treatment. Scale bar, 10 μm. Data are presented as mean ± SEM of 3 independent experiments. *P < 0.05, **P < 0.01.