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

Journal: Cell reports

Article Title: Proximity proteomics reveals UCH-L1 as an essential regulator of NLRP3-mediated IL-1β production in human macrophages and microglia.

doi: 10.1016/j.celrep.2024.114152

Figure Lengend Snippet: Figure 6. UCH-L1 interacts with the NACHT domain of NLRP3 (A) The raw label-free quantitation (LFQ) intensity of UCH-L1 protein identified in the APEX2 PL performed in this study across all samples, including bead control, DMSO control, and nigericin treatment at different time points. The experiment was performed in triplicate except for Nig-30 and Nig-60, where only 1 replication was performed. (B) Flp-In T-Rex 293 NLRP3-FLAG stable cells were induced with tetracycline overnight. Afterward, cells were treated with DMSO or nigericin for 90 min. NLRP3- FLAG was pulled down using anti-FLAG antibody or immunoglobulin G (IgG) and immunoblotted with the indicated antibodies. (C) BV2 microglia cells were treated with or without LPS (1 mg/mL) for 4 h, followed by co-immunoprecipitation with anti-NLRP3 antibody or isotype control and analysis by immunoblotting. (D) Schematic of the N-terminal FLAG-tagged NLRP3 domain constructs: full-length NLRP3, NACHT and LRR domain NLRP3 (FLAG-NACHT+LRR, amino acids 140–1,034), LRR domain of NLRP3 (FLAG-LRR, amino acids 700–1,034), pyrin and NACHT domain of NLRP3 (FLAG-PYD+NACHT, amino acids 1–700), and NACHT domain of NLRP3 (FLAG-NACHT, amino acids 140–700). (E) HEK293T cells were transfected with the appropriate NLRP3 plasmid using Lipofectamine 3000. Cells were cultured for 24 h before they were lysed. NLRP3 domain-FLAG was pulled down using anti-FLAG antibody and immunoblotted with the indicated antibodies. (F) Fluorescence confocal images of hiPSC-derived microglial cells stained for UCHL-1 and NLPR3. UCHL-1 and NLRP3 show colocalization, but only when cells are stained with primary antibodies for UCHL-1 and NLRP3 (right column). No colocalization is shown when cells are not stained with primary antibodies against UCHL-1 and NLRP3 (left column). (G) Line profile showing the min-max normalized intensity of UCHL-1 (red) and NRLP3 (green) along and in the direction of the white arrow indicated in (D). The peaks in normalized intensity indicate colocalization of NLRP3 and UCHL-1 in enriched areas (or ‘‘specks’’) of NLRP3.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Flp-In T-REx 293 Cell Line ThermoFisher Scientific CAT# R78007 Recombinant DNA pOG44 Plasmid Vector ThermoFisher Scientific CAT# V600520 pcDNA5/FRT/TO GFP Addgene CAT# 19444 pcDNA5/FRT/TO APEX2-MyD88-Flag Generated in our Lab (Backbone 19444) N/A pcDNA5/FRT/TO APEX2-NLRP3-Flag Generated in this study (Backbone 19444) N/A pEGFP-C2-NLRP3 Addgene CAT# 73955 pNLRP3 FLAG Full-length Generated in this study (Backbone 73955) N/A pNLRP3 NACHT+ LRR- FLAG (140–1035) Generated in this study (Backbone 73955) N/A pNLRP3 LRR only- FLAG (700–1035) Generated in this study (Backbone 73955) N/A pNLRP3 Pyrin + NACHT- FLAG (1–700) Generated in this study (Backbone 73955) N/A pNLRP3 NACHT only- FLAG (140–700) Generated in this study (Backbone 73955) N/A lentiCRISPR v2 Addgene CAT# 52961 lentiCRISPRv2 with UCH-L1-sgRNA#1 Generated in this study (Backbone lentiCRISPRv2) N/A lentiCRISPRv2 with UCH-L1-sgRNA#2 Generated in this study (Backbone lentiCRISPRv2) N/A lentiCRISPRv2 with UCH-L1-sgRNA#3 Generated in this study (Backbone lentiCRISPRv2) N/A Software and algorithms Prism, version 10.1.2 GraphPad https://www.graphpad.com/ scientific-software/prism/ Perseus, version 2.0.7.0 Tyanova et al.65 https://maxquant.net/perseus/ Image Studio Lite, version 5.2 LI-COR https://www.licor.com/bio/ image-studio-lite/ FiJi Schindelin et al.66 https://imagej.net/software/fiji/ Cytoscape, version 3.9.0 Shannon et al.67 https://cytoscape.org ColumbusTM Image Data Storage and Analysis system, version 2.9.0 Revvity https://www.revvity.com Illustrator Adobe https://www.adobe.com Photoshop Adobe https://www.adobe.com MaxQuant, Version v1.6.10.43 Tyanova et al.68 http://www.maxquant.org 20 Cell Reports 43, 114152, May 28, 2024 d This study does not report original code. d Any additional information required to reanalyse the data reported in this paper is available from the lead contact upon request.

Techniques: Quantitation Assay, Control, Immunoprecipitation, Western Blot, Construct, Transfection, Plasmid Preparation, Cell Culture, Fluorescence, Derivative Assay, Staining