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. 2024 Feb 19;12:RP88686. doi: 10.7554/eLife.88686

Figure 2. NAD+ specifically inhibits the non-canonical inflammasome by targeting caspase-11.

Bone marrow was isolated from mice and bone marrow-derived macrophages (BMDMs) were differentiated in vitro. Subsequently, BMDMs were cultured in the presence of NAD+ or PBS. BMDMs were then primed with either Pam3CSK4 or lipopolysaccharide (LPS) O111:B4. Next primed BMDMs were stimulated with ATP or LPS and cholera toxin B (CTB). (A) Pro-casp-1, pro-casp-11, casp-11, NLRP3, casp-1, IL1β, and gasdermin D (GSDMD) expression were determined using western blot and (B) IL-1β secretion and LDH release were assessed in the supernatant. Column plots display mean with standard deviation (n=5-8). (C) Time-dependent caspase-1 expression was determined via active staining and assessed using a confocal microscope. Column plots display mean with standard deviation (n=5) (D) Cell viability and apoptosis were monitored using the IncuCyte live microscopy system. (E) LPS transfection with CTB was visualized by using FITC-coupled LPS and DAPI staining and quantified by confocal microscopy and flow cytometry. Column plots display mean with standard deiation (n=6) (F) For human experiments macrophages were differentiated from PBMC, primed with Pam3CSK4 and subsequently transfected with LPS and 0.25% Fugene HD Plus. Column plots display mean with standard deviation (n=6). Statistical significance was determined by using Student’s t-test or one-way ANOVA. Asterisks indicate p-values *=p<0.05, **=p<0.01, and ***=p<0.001, only significant values are shown. All data depicted in this figure are provided as source data.

Figure 2—source data 1. Raw data for Figure 2A: Original western blots.
Figure 2—source data 2. Raw data for Figure 2A: Western blots with highlighted bands and sample labels.
Figure 2—source data 3. Raw data for Figure 2B: ELISA mouse bone marrow-derived macrophages (BMDMs).
Figure 2—source data 4. Raw data for Figure 2C: Caspase-1 staining.
Figure 2—source data 5. Raw data for Figure 2D: IncuCyte live microscopy.
Figure 2—source data 6. Raw data for Figure 2E: Lipopolysaccharide (LPS) transfection staining.
Figure 2—source data 7. Raw data for Figure 2F: ELISA human macrophages.

Figure 2.

Figure 2—figure supplement 1. NAD+ does not alter bone marrow-derived macrophage (BMDM)-derived NF-κB expression or phosphorylation.

Figure 2—figure supplement 1.

Differentiated BMDMs were cultured in the presence of 100 µmol NAD+ or PBS for 2 following days. BMDMs were then primed with 1 µg/ml Pam3CSK4 and subsequently stimulated with 2 µg/ml lipopolysaccharide (LPS) O111:B4 and 20 µg/ml cholera toxin B (CTB). Unstimulated BMDMs served as controls. (A) P52 and p65 expression was determined using western blot. (B) Stimulated BMDMs were stained with p52, p65, and phospho-p65 and expression levels assessed using confocal microscopy. All data depicted in this figure are provided as source data.
Figure 2—figure supplement 1—source data 1. Raw data for Figure 2—figure supplement 1A: Western blot.
Figure 2—figure supplement 1—source data 2. Raw data for Figure 2—figure supplement 1A: Western blots bands with highlighted and sample labels.
Figure 2—figure supplement 1—source data 3. Raw data for Figure 2—figure supplement 1B: Immunofluorescence.
Figure 2—figure supplement 2. Unstimulated bone marrow-derived macrophage (BMDM) cell viability and apoptosis.

Figure 2—figure supplement 2.

Differentiated BMDMs were cultured in the presence of 100 µmol NAD+ or PBS for 2 following days. BMDMs were then primed with 1 µg/ml Pam3CSK4, subsequently stimulated with 2 µg/ml lipopolysaccharide (LPS) O111:B4 and 20 µg/ml cholera toxin B (CTB), and cell viability and apoptosis were monitored for 100 hr using the IncuCyte live microscopy system. All data depicted in this figure are provided as source data.
Figure 2—figure supplement 2—source data 1. Raw data for Figure 2—figure supplement 2: IncuCyte live microscopy.