rabbit α mouse caspase 11  (R&D Systems)

Journal: eLife

Article Title: NAD + prevents septic shock-induced death by non-canonical inflammasome blockade and IL-10 cytokine production in macrophages

doi: 10.7554/eLife.88686

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

Article Snippet: The following primary antibodies were used according to the manufacturer’s instructions: pro-caspase-1 (#ab179515, Abcam), caspase-1 (#14-9832-82, eBioscience), IL-1β (AF-401-NA, R&D Systems), NLRP3 (#768319, R&D Systems), caspase-11 (#mab8648, R&D Systems), GSDMD (ab209845, Abcam), P-STAT-1 (#9167S, Cell Signaling), STAT-1 (#9172S, Cell Signaling), NF-κB-p65 (#49445S, Cell Signaling), NF-κB-p52 (#4882S, Cell Signaling), β-actin (ab3280, Abcam).

Techniques: Isolation, Derivative Assay, In Vitro, Cell Culture, Expressing, Western Blot, Standard Deviation, Staining, Microscopy, Transfection, Confocal Microscopy, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: eLife

Article Title: NAD + prevents septic shock-induced death by non-canonical inflammasome blockade and IL-10 cytokine production in macrophages

doi: 10.7554/eLife.88686

Figure Lengend Snippet: Differentiated bone marrow-derived macrophages (BMDMs) were cultured in the presence of NAD + or PBS. BMDMs were then primed with Pam3CSK4, subsequently stimulated with lipopolysaccharide (LPS) and cholera toxin B (CTB) and RNA-sequencing was performed. Unstimulated BMDMs served as controls. ( A ) Venn diagram plotting common gene expression between all three groups. ( B ) Gene ontology enrichment analysis displaying the highest significant pathways differing when comparing NAD + and PBS-treated BMDMs. ( C ) Expression cluster analysis of genes involved in IFN-β signaling through cluster analysis depicted in a heat map. ( D ) Volcano plot displaying the most significant genes up- or downregulated comparing NAD + and PBS-treated BMDMs. ( E ) Stimulated BMDMs were additionally treated with recombinant INF-β, and IL-1β and LDH release were measured. Column plots display mean with standard deviation (n=6) ( F ) Moreover, pro-casp-1, casp-11, NLRP3, gasdermin D (GSDMD), ( G ) signal transducer activator of transcription-1 (STAT-1), and phospho-STAT-1 expression were assessed by western blot. 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 3—source data 1. Raw data for : ELISA bone marrow-derived macrophage (BMDM). Figure 3—source data 2. Raw data for : Original western blots. Figure 3—source data 3. Raw data for : Western blots with highlighted bands and sample labels. Figure 3—source data 4. Raw data for : Original western blots. Figure 3—source data 5. Raw data for : Western blots with highlighted bands and sample labels.

Article Snippet: The following primary antibodies were used according to the manufacturer’s instructions: pro-caspase-1 (#ab179515, Abcam), caspase-1 (#14-9832-82, eBioscience), IL-1β (AF-401-NA, R&D Systems), NLRP3 (#768319, R&D Systems), caspase-11 (#mab8648, R&D Systems), GSDMD (ab209845, Abcam), P-STAT-1 (#9167S, Cell Signaling), STAT-1 (#9172S, Cell Signaling), NF-κB-p65 (#49445S, Cell Signaling), NF-κB-p52 (#4882S, Cell Signaling), β-actin (ab3280, Abcam).

Techniques: Derivative Assay, Cell Culture, RNA Sequencing, Gene Expression, Expressing, Recombinant, Standard Deviation, Western Blot, Enzyme-linked Immunosorbent Assay