Fig 2: SIRT1 knockdown enhanced the activation of NLRP3 inflammasome. (a) The SIRT1 knockdown lentiviral vector (LV) was transfected into BV2 microglial cells. The expression of EGFP was observed by fluorescence microscope; (b) the mRNA of SIRT1 of SIRT1 knockdown cells was determined by RT–PCR; (c) the protein expression of SIRT1 of SIRT1 knockdown cells was determined by western blotting; (d) the quantitative blots of SIRT1; (e) cells were divided into four groups: control, control+10 µg/ml LPS+4 mM ATP, LV-NC+10 µg/ml LPS+4 mM ATP, and LV-down+10 µg/ml LPS+4 mM ATP group. The protein expression of SIRT1, NLRP3, ASC, and IL-1ß was detected by western blotting; (f) the quantitative blots of SIRT1; (g) the quantitative blots of NLRP3, ASC, and IL-1ß; (h) the gene expression of SIRT1; (i) the gene expression of IL-1ß. *P < 0.05, compared with the control group. #P < 0.05, compared with the LV-NC+10 µg/ml LPS+4 mM ATP group. n = 5 per group. The data represent the means ± SD.

Fig 3: NLRP3 inflammasome activation in non-RPE cells but not in RPE cells promotes CNV.(A) Nuclear Cre immunostaining (white) in choroidal flat mounts from 6-weeks-old VegfahyperBest1Cre/+Nlrp3A350V/A350V mice confirms uniform Cre expression in the RPE. Inset shows higher magnification image. No Cre staining is seen in VegfahyperCrenegNlrp3A350V/A350V mice ( = VegfahyperNlrp3-/- mice). Scale bars, 200 μm. (B-C). CNV lesion numbers and average CNV lesion area (in μm2/lesion) were measured in 6-weeks-old mice. VegfahyperNlrp3-/- mice (group 2) and VegfahyperBest1Cre/+Nlrp3A350V/A350V mice (group 3) show significantly decreased CNV lesion numbers compared to Vegfahyper mice (group 1), but no significant difference in CNV lesion sizes. CNV lesion sizes and numbers between VegfahyperNlrp3-/- mice and VegfahyperBest1Cre/+Nlrp3A350V/A350V mice are similar. No marked differences in lesion sizes or lesion numbers were observed among male or female groups of VegfahyperNlrp3-/- mice and VegfahyperBest1Cre/+Nlrp3A350V/A350V mice. VegfahyperCasp1-/-Casp11-/- mice (group 4) have fewer CNV lesions than VegfahyperNlrp3-/- mice or VegfahyperBest1Cre/+Nlrp3A350V/A350V mice. Values represent total CNV lesion numbers/eye for each mouse and average lesion area/eye for each mouse. Absolute numbers of mice per group are indicated in parentheses. Graphs show mean ± SEM. P-values are shown and were determined by a Kruskal-Wallis test followed by a Dunn’s test.

Fig 4: Ex vivo heart perfusion combined with melatonin post-conditioning exerts a profound cardioprotective effect against myocardial IRI in the DCD heart, and this protection appears to be due to the inhibition of NLRP3 inflammasome-mediated pyroptosis. IRI, ischemia/reperfusion injury; DCD, donation after circulatory death.

Fig 5: Pathogenicity validation of novel TNFAIP3 variants. (A) The predicted 3D structure of wild type (WT) and A547T variant of A20 protein. (B) Schematic view of Complementary DNA analysis of WT and the splice site mutation c.1906+2T>G. (C) Agarose gel electrophoresis result. P2, the proband of the second family; Ctrl, healthy controls. (D) Western blot analysis of A20 expression, NF-κB and NLRP3 inflammasome signaling pathway in PBMCs from P2 and controls. (E) Cytokine (TNF-α, IL-6 and IL-1β) levels in plasma and culture supernatants of PBMCs from P2 and controls (mean ± SD from three independent experiments in P2 and n=3 controls, P values were determined by unpaired two-tailed Student’s t-test).