Fig. 6: The phosphorylation of NCF4 is important for NLRP3 inflammasome activation. | Nature Communications

Fig. 6: The phosphorylation of NCF4 is important for NLRP3 inflammasome activation.

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

Fig. 6

a, b WT and Ncf4–/– BMDMs were pretreated with PKC412 (25 nm, 50 nm, and 100 nm) for 1 h, and further stimulated with LPS (500 ng/mL, 4 h) plus ATP (5 mM, 60 min) for NLRP3 inflammasome activation. Immunoblot analysis of pro-caspase-1 (Pro-Casp1) and its subunit p20 (a), and analysis of IL-1β release (b, n = 4 biologically independent samples) in PKC412-treated and untreated BMDMs with and without treatment for NLRP3 inflammasome activation. c Immunoblot analysis of phosphorylation of NCF4, pro-caspase-1 (Pro-Casp1) and its subunit p20 in untreated and PKC412-treated WT BMDMs with and without treatment with the NLRP3 activator LPS plus ATP (LPS, 500 ng/mL, 4 h and ATP, 5 mM, 60 min) or the NLRC4 activator Salmonella enterica Typhimurium (3 MOI, 2 h). d, e Immunoblot analysis of pro-caspase-1 (Pro-Casp1) and its subunit p20 (d), and analysis of IL-1β release (e, n = 4 biologically independent samples) in Ncf4–/– BMDMs transduced with WT NCF4 and single or quadruple mutations (NCF4MT) of phosphorylation sites of NCF4 as indicated, further stimulated with LPS plus ATP (LPS, 500 ng/mL, 4 h and ATP, 5 mM, 60 min). f Co-IP analysis of transduced NCF4 interacting with ASC and NOX2 in Ncf4–/– BMDMs transduced with control plasmid (PCDH), WT NCF4 and quadruple mutations (NCF4MT) of phosphorylation sites of NCF4 without treatment (Media), treated with LPS (500 ng/mL, 4.5 h) or with LPS plus ATP (LPS, 500 ng/mL, 4 h and ATP, 5 mM, 15 min). g ASC oligomerization analysis in Ncf4–/– BMDMs transduced with control plasmid (PCDH), WT NCF4, and quadruple mutations (NCF4MT) of phosphorylation sites of NCF4 without treatment (Media) and treated with LPS plus ATP (LPS, 500 ng/mL, 4 h and ATP, 5 mM, 30 min). h Confocal microscopy analysis NCF4, NCF4MT, NLRP3, and ASC subcellular localization in Ncf4–/– BMDMs transduced with control plasmid (PCDH), WT NCF4 and quadruple mutations (NCF4MT) of phosphorylation sites of NCF4 response to LPS plus ATP (LPS, 500 ng/mL, 4 h and ATP, 5 mM, 30 min). Scale bars: 20 μm. Data are from 3 (b, e) or representative of 3 independent experiments with similar results (a, c, d, f–h). Data represent Mean ± SEM for (b, e), 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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