Supplementary Fig. 4
Deficiency of diacylglycerol acyltransferase-1 (DGAT1) suppresses nucleotide-binding domain, leucine-rich-repeat-containing receptor (NLR), pyrin-domain-containing 3 (NLRP3) inflammasome activation. (A) Representative immunoblot analysis for DGAT1, caspase-1, and interleukin 1β (IL-1β; left), and densitometry quantification of DGAT1, caspase-1 p10 and IL-1β p17 levels (normalized to levels of β-actin; right) from wild-type (WT) bone marrow-derived macrophages (BMDMs) were transduced with Dgat1-targeting gRNAs (Dgat1 gRNA #1 and Dgat1 gRNA #2), or with a control plasmid (control), and were stimulate with nigericin after lipopolysaccharide (LPS) incubation. (B) Quantification of IL-1β (left), IL-18 (middle), and tumor necrosis factor α (TNF-α; right) secretion from WT BMDMs were transduced with Dgat1-targeting gRNAs (Dgat1 gRNA #1 and Dgat1 gRNA #2), or with a control plasmid (control), and were stimulated with nigericin or adenosine triphosphate (ATP) after LPS incubation. All data are mean±standard deviation. Data are representative of three independent experiments and each carried out in triplicate. aP<0.01, bP<0.05, by two-tailed t-test.
dmj-43-683-s004.pdf
Fig. 1PF-04620110 suppresses fatty acid-induced nucleotide-binding domain, leucine-rich-repeat-containing receptor (NLR), pyrin-domain-containing 3 (NLRP3) inflammasome activation. (A) Quantification of interleukin 1β (IL-1β; left), IL-18 (middle), and tumor necrosis factor α (TNF-α; right) secretion from wild-type (WT) bone marrow-derived macrophages (BMDMs) were pretreated with PF-04620110 (50 µM, 2 hours) or dimethyl sulfoxide (DMSO) (control), followed by incubation with palmitate-bovine serum albumin (PA-BSA) after lipopolysaccharide (LPS) stimulation (n=10 mice per group). (B) Quantification of IL-1β secretion from WT BMDMs that were pretreated with PF-04620110 in a dose-dependent manner (12.5, 25, 50, or 100 µM, 2 hours) or DMSO (control), followed by incubation with PA-BSA after LPS stimulation (n=10 mice per group). (C) Quantification of IL-1β secretion from WT BMDMs that were pretreated with PF-04620110 (50 µM, 2 hours) or DMSO (control), followed by incubation with poly(dA:dT) or flagellin after LPS stimulation (n=10 mice per group). (D) Representative immunoblot analysis for caspase-1 and IL-1β (left), and densitometry quantification of caspase-1 p10 and IL-1β p17 levels (normalized to levels of β-actin) (right), from WT BMDMs that were pretreated with PF-04620110 (50 µM, 2 hours) or DMSO, followed by incubation with PA-BSA after LPS stimulation. For immunoblots, β-actin was used as loading control (n=6 mice per group). Data are mean±standard deviation. aP<0.001, bP<0.01, cP<0.05 by two-tailed t-test or analysis of variance.
Fig. 2PF-04620110 inhibits K+ efflux and the formation of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) specks during nucleotide-binding domain, leucine-rich-repeat-containing receptor (NLR), pyrin-domain-containing 3 (NLRP3) inflammasome activation. (A) Quantification of triglyceride levels from wild-type (WT) bone marrow-derived macrophages (BMDMs) that were pretreated with PF-04620110 (50 µM, 2 hours) or dimethyl sulfoxide (DMSO; control), followed by incubation with palmitate-bovine serum albumin (PA-BSA) after lipopolysaccharide (LPS) stimulation (n=10 mice per group). (B) Quantification of triglyceride levels from WT BMDMs that were pretreated with PF-04620110 in a dose-dependent manner (12.5, 25, 50, or 100 µM, 2 hours) or DMSO (control), followed by incubation with PA-BSA after LPS stimulation (n=10 mice per group). (C) Intracellular Ca2+ flux assays from WT BMDMs that were pretreated with PF-04620110 (50 µM, 2 hours) or DMSO (control), followed by incubation with PA-BSA after LPS stimulation (n=3 mice per group). (D) Quantification of interleukin 1β (IL-1β; left), IL-18 (middle), and tumor necrosis factor α (TNF-α; right) secretion from WT BMDMs that were pretreated with PF-04620110 (50 µM, 2 hours), KCl (100 mM, 1 hour), or DMSO (control), followed by incubation with PA-BSA after LPS stimulation (n=6 mice per group). (E) Representative immunoblot analysis for caspase-1 and IL-1β (left), and densitometry quantification of caspase-1 p10 and IL-1β p17 levels (normalized to levels of β-actin) (right), from WT BMDMs that were pretreated with PF-04620110 (50 µM, 2 hours), KCl (100 mM, 1 hour), or DMSO, followed by incubation with PA-BSA after LPS stimulation. For immunoblots, β-actin was used as loading control (n=6 mice per group). (F) Representative immunofluorescence images (total 100 cells in 15 individual images per group; left), and quantification (right), of ASC speck formation (white arrows) (the percent of ASC speck positive cells for each mouse) in WT BMDMs that were pretreated with PF-04620110 (50 µM, 2 hours) or DMSO, followed by incubation with adenosine triphosphate (ATP) after LPS stimulation. Scale bars, 20 µm (n=5 mice per group). Data are mean±standard deviation. Data are representative of three independent experiments, and each was done in triplicate. aP<0.001, bP<0.01, cP<0.05 by two-tailed t-test or analysis of variance.
Fig. 3Genetic inhibition of diacylglycerol acyltransferase-1 (DGAT1) suppresses fatty acid-induced nucleotide-binding domain, leucine-rich-repeat-containing receptor (NLR), pyrin-domain-containing 3 (NLRP3) inflammasome activation. (A) Representative immunoblot analysis for DGAT1, caspase-1, and interleukin 1β (IL-1β; left), and densitometry quantification of DGAT1, caspase-1 p10, and IL-1β p17 levels (normalized to levels of β-actin; right), from wild-type (WT) bone marrow-derived macrophages (BMDMs) were transduced with two independent Dgat1-targeting gRNAs (Dgat1 gRNA #1 and Dgat1 gRNA #2), or with a control plasmid (control), and were stimulated with lipopolysaccharide (LPS) and palmitate-bovine serum albumin (PA-BSA). For immunoblots, β-actin was used as loading control (n=5 mice per group). (B) Quantification of IL-1β (left), IL-18 (middle), and tumor necrosis factor α (TNF-α; right) secretion from WT BMDMs were transduced with two independent Dgat1-targeting gRNAs (Dgat1 gRNA #1 and Dgat1 gRNA #2), or with a control plasmid (control), and were stimulated with LPS and PA-BSA (n=10 mice per group). (C) Quantification of IL-1β and IL-18 secretion from WT BMDMs that were transduced with two independent Dgat1-targeting gRNAs (Dgat1 gRNA #1 and Dgat1 gRNA #2), or with a control plasmid (control), and were incubated with poly(dA:dT) or flagellin after LPS stimulation (n=10 mice per group). Data are mean±standard deviation. aP<0.01, bP<0.05, by two-tailed t-test or analysis of variance.
Fig. 4Deficiency of diacylglycerol acyltransferase-1 (DGAT1) suppressed K+ efflux and the formation of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) specks during nucleotide-binding domain, leucine-rich-repeat-containing receptor (NLR), pyrin-domain-containing 3 (NLRP3) inflammasome activation. (A) Quantification of triglyceride levels from wild-type (WT) bone marrow-derived macrophages (BMDMs) that were transduced with Dgat1-targeting gRNAs (Dgat1 gRNA), or with a control plasmid (control), and that were stimulated with lipopolysaccharide (LPS) and palmitate-bovine serum albumin (PA-BSA) (n=10 mice per group). (B) Intracellular Ca2+ flux assays from WT BMDMs that were transduced with Dgat1-targeting gRNAs (Dgat1 gRNA), or with a control plasmid (control), and that were stimulated with PA-BSA stimulation after LPS incubation (n=3 mice per group). (C) Representative immunofluorescence images (total 100 cells in 15 individual images per group) (left), and quantification (right), of ASC speck formation (white arrows) (the percent of ASC speck positive cells for each mouse) in WT BMDMs that were transduced with Dgat1-targeting gRNAs (Dgat1 gRNA), or with a control plasmid (control), and that were stimulated with LPS and PA-BSA. Scale bars, 20 µm (n=5 mice per group). Data are mean±standard deviation. aP<0.01, by two-tailed t-test or analysis of variance.
Fig. 5PF-04620110 suppressed high-fat diet (HFD)-induced interleukin 1β (IL-1β) and IL-18 production in mice. Quantification of (A) IL-1β (left), (B) IL-18 (right) levels in plasma from wild-type (WT) mice fed HFD or regular diet (RD) for 12 weeks, and then treated with PF-04620110 or vehicle control (dimethyl sulfoxide [DMSO]) once a day at the dose of 3 mg/kg for another 4 weeks (RD n=5, HFD n=10; RD+PF-04620110 n=5, HFD+PF-04620110 n=10). (C) Fasting and (D) fed blood glucose levels from WT mice fed HFD or RD for 12 weeks, and then treated with PF-04620110 or vehicle control (DMSO) once a day at the dose of 3 mg/kg for another 4 weeks (RD n=5, HFD n=10, HFD+PF-04620110 n=10). Data are mean±standard deviation. aP<0.01, bP<0.05, by two-tailed t-test or analysis of variance.