Skip to main content
. 2023 Jul 21;4(8):101129. doi: 10.1016/j.xcrm.2023.101129

Figure 6.

Figure 6

Phenylpyruvate increased NLRP3 protein stability and promoted inflammasome activation

(A) Immunoblot assay and statistical analysis showing the stability of NLRP3 protein in the indicated cells. BMDMs were treated with CHX (100 μg/mL) for 0, 3, 6, and 9 h and then collected for immunoblot analysis (n = 3).

(B) Immunoblot analysis showing NLRP3 protein levels in BMDMs treated with proteasome inhibitors MG132 (10 μM), carfilzomib (100 nM), or autophagy and autolysosome inhibitors Baf A1 (0.2 μM) or CQ (50 μM) for 8 h (n = 3).

(C) Alignment of NLRP3 pyrin domain-containing palmitoylation sites among different species.

(D) BMDMs were treated with 400 μM phenylpyruvate for 4 h and LPS (100 ng/mL) for 24 h and then stimulated with ATP (3 mM) for 45 min. Cell lysates were collected to determine the interaction between NLRP3 and ASC by IP and immunoblot analysis (n = 3).

(E) Arrows showing ASC immunofluorescence in BMDMs treated with phenylpyruvate (400 μM) for 4 h and LPS (100 ng/mL) for 24 h, followed by stimulation with ATP (3 mM) for 45 min. Scale bar, 50 μm.

(F) Immunoblot analysis of the supernatant and cell extracts in the indicated BMDMs, which were treated with phenylpyruvate for 4 h and LPS for 24 h, with transfection of Ppt1 WT or MUT plasmid, followed by stimulation with ATP for 45 min (n = 3).

(G–I) BMDMs were treated with phenylpyruvate (400 μM) for 4 h and LPS for 24 h, with transfection of Ppt1 WT or MUT plasmid, followed by stimulation with ATP for 45 min. Levels of the inflammatory factors IL-1β, IL-18, and TNFα in the supernatant were measured using ELISA (n = 3).

(J–L) BMDMs were treated with phenylpyruvate (400 μM) for 4 h and LPS for 24 h and transfected with the Ppt1 WT or MUT plasmid. The RT-qPCR results showing relative mRNA expression of Nos, Tnf, and Il6 normalized to Actb (n = 3).

(M–O) RT-qPCR analysis of relative mRNA expression of Nos, Tnf, and Il6 when giving phenylpyruvate (400 μM) to either NLRP3 inhibitor MCC950-treated (1 μM) or NLRP3-knockdown BMDMs (n = 3). Data are shown as mean ± SD. ∗p < 0.05, ∗∗p < 0.01; n.s., not significant.