Fig. 2: NCF4 mediates the activation of both NLRP3 and AIM2 inflammasomes. | Nature Communications

Fig. 2: NCF4 mediates the activation of both NLRP3 and AIM2 inflammasomes.

From: NCF4 attenuates colorectal cancer progression by modulating inflammasome activation and immune surveillance

Fig. 2

a–c Immunoblot analysis of pro-caspase-1 (Pro-Casp1), its subunit p20, total and phosphorylated NCF4 in WT and Ncf4–/– BMDMs without treatment (Med) or stimulated with LPS (500 ng/mL, 4 h) and ATP (5 mM, 30 min and 60 min) for NLRP3 inflammasome activation (a), transfected with dsDNA (1.5 μg, 1 h and 2 h) or infected with F. novicida (200 MOI, 12 h and 24 h) for AIM2 inflammasome activation (b), and Salmonella enterica Typhimurium (3 MOI, 2 h and 4 h) for NLRC4 inflammasome activation (c). d Analysis of IL-1β release in WT and Ncf4–/– BMDMs without treatment (Media) or stimulated with LPS (500 ng/mL, 4 h) and ATP (5 mM, 60 min), transfected with dsDNA (1.5 μg, 2 h), infected with F. novicida (200 MOI, 24 h), and Salmonella enterica Typhimurium (3 MOI, 4 h) (n = 4 biologically independent samples). e Confocal microscopy analysis of ASC speck formation in WT and Ncf4–/– BMDMs without treatment or stimulated with LPS (500 ng/mL, 4 h) and ATP (5 mM, 30 min), or transfected with dsDNA (1.5 μg, 1 h); or infected with F. novicida (200 MOI, 12 h) as indicated. Arrows indicate ASC specks. Scale bars: 10 μm. f–h WT and Ncf4–/– BMDMs were transfected with siRNAs of Ncf1 and Ncf2, and further treated with LPS (500 ng/mL, 4 h) and ATP (5 mM, 60 min), and transfected with dsDNA (1.5 μg, 2 h) for inflammasome activation analysis. f qRT-PCR analysis of Ncf1, Ncf2, and Ncf4 in WT and Ncf4–/– BMDMs transfected with control siRNA or siRNAs of Ncf1 and Ncf2 (n = 4 technical replicates; 3 independent experiments). g, h Immunoblot analysis of pro-caspase-1 (Pro-Casp1), its subunit p20, NCF4, NCF2, and NCF1 (g), and analysis of IL-1β release (h) in WT and Ncf4–/– BMDMs transfected with siRNAs and further treated with inflammasome stimuli as indicated (n = 4 biologically independent samples). Data are from 3 (d, h) or representative of 3 independent experiments with similar results (a–c, e–g). Data represent Mean ± SEM for (d, f, h), 2-sided Student’s t-test without multiple-comparisons correction, p-value is indicated in the graph. Source data are provided as a Source Data file.

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