Journal: Cell

Article Title: NLRC5 senses NAD + depletion to form a PANoptosome driving PANoptosis and inflammation

doi: 10.1016/j.cell.2024.05.034

Figure Lengend Snippet: (A) Wild-type (WT) and Nlrc5−/− bone marrow-derived macrophages (BMDMs) were either left unstimulated (0 h) or treated with heme plus Pam3CSK4 (Pam3) for 36 h and stained for NLRC5, and counter-stained with DAPI to visualize nuclei. A broader, enlarged field of view for WT BMDMs at the 36 h timepoint is shown. (B) Western blot analysis of NLRC5 in WT BMDMs stimulated with heme plus Pam3 for the indicated times. For loading control, β-actin is shown. (C) WT and Nlrc5−/− BMDMs were either unstimulated (0 h) or treated with heme plus Pam3 for 36 h and stained for NLRC5, ASC, caspase-8 (CASP8), and RIPK3, and counter-stained with DAPI to visualize nuclei. Representative images of cells containing co-localized NLRC5, ASC, CASP8, and RIPK3 are shown. The magnified view of the boxed area (merged) is shown on the right (enlarged). (D) Quantification showing the percentage of cells with NLRC5+ASC+ CASP8+ RIPK3+ specks out of the total population of cells with ASC+ specks in WT and Nlrc5−/− BMDMs at 36 h post-stimulation with heme plus Pam3. Each data point indicates a single field of view (average n = 103 cells per field). (E) Immunoblot analysis (IB) of ASC, NLRC5, NLRP3, CASP8, and RIPK3 following immunoprecipitation (IP) with IgG control or anti-ASC antibodies in WT, Nlrp12−/−, Pycard−/−, and Nlrc5−/− BMDMs after treatment with heme plus Pam3 (H+P) for 28 h. Scale bars = 10 μm (A), 5 μm (C, merge column), and 1 μm (C, enlarged column). Nlrc5−/− BMDMs (ref. 56) (A, C–E) were used for stimulation. Three or more independent experiments were performed, and the data shown are from a single experiment that is representative. Mean ± SEM are shown (D). Statistical analyses were performed using the unpaired t test (D). ****P < 0.0001. See also Figure S5.

Article Snippet: For immunoprecipitation in the overexpression system, HEK293T cells (CRL-3216, ATCC) were seeded into six-well plates and transfected with a combined total of 5 ug of the following plasmids (1:1:1:1:1, molar ratio): pcDNA3-Myc-h ASC (#73952, Addgene), pcDNA3-h CASP8 (#11817, Addgene), pcDNA3-HA-h RIPK3 (#78804, Addgene), pcDNA3-Myc-h NLRC5 (#37509, Addgene), and pcDNA3-h NLRP12 -FLAG (Human NLRP12 PCR product amplified using cDNA and cloned in pcDNA TM 3.1 (+) Mammalian Expression Vector (#V790-20, Thermo Fisher Scientific), with or without an equimolar ratio of pEGFP-C2-h NLRP3 (#73955, Addgene), and incubated for 24–36 h. Immunoprecipitation was performed as previously described 12 , 102 with minor modifications.

Techniques: Derivative Assay, Staining, Western Blot, Control, Immunoprecipitation

Journal: Cell

Article Title: NLRC5 senses NAD + depletion to form a PANoptosome driving PANoptosis and inflammation

doi: 10.1016/j.cell.2024.05.034

Figure Lengend Snippet: Key Resources Table

Article Snippet: For immunoprecipitation in the overexpression system, HEK293T cells (CRL-3216, ATCC) were seeded into six-well plates and transfected with a combined total of 5 ug of the following plasmids (1:1:1:1:1, molar ratio): pcDNA3-Myc-h ASC (#73952, Addgene), pcDNA3-h CASP8 (#11817, Addgene), pcDNA3-HA-h RIPK3 (#78804, Addgene), pcDNA3-Myc-h NLRC5 (#37509, Addgene), and pcDNA3-h NLRP12 -FLAG (Human NLRP12 PCR product amplified using cDNA and cloned in pcDNA TM 3.1 (+) Mammalian Expression Vector (#V790-20, Thermo Fisher Scientific), with or without an equimolar ratio of pEGFP-C2-h NLRP3 (#73955, Addgene), and incubated for 24–36 h. Immunoprecipitation was performed as previously described 12 , 102 with minor modifications.

Techniques: Control, Virus, Recombinant, In Vitro, In Vivo, Protease Inhibitor, Western Blot, Iron Assay, AST Assay, Magnetic Beads, Enzyme-linked Immunosorbent Assay, Isolation, Transfection, Antibody Labeling, Gene Expression, Software