Journal: Developmental cell

Article Title: In vivo developmental trajectories of human podocyte development inform in vitro differentiation of pluripotent stem-cell derived podocytes

doi: 10.1016/j.devcel.2019.06.001

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Primary antibodies used in the study are listed as follow: LHX1 (R&D, MAB2725,1:300), MAFB (R&D, MAB3810, 1:500), MAFB (Santa Cruz, sc-10022, 1:100), PAX8 (abcam, ab189249, 1:1000), KRT8/18 (DSHB, troma-1; 1:50), NPHS2 (abcam, ab50339, 1:10000), SYNPO (R&D, MAB8977, 1:300), TGFBR3 (R&D, AF-242-PB, 1:300), ANXA1 (Cell Signaling, 32934, 1:200), ARMH4/C14orf37 (Sigma Aldrich, HPA001789, 1:300), WT1 (abcam, ab89901, 1:1000), CUBN (Santa Cruz, sc-20607, 1:300), CDH1(Biosciences, 610182, 1:300 ), PDGFRB (abcam, ab32570, 1:500), VEGFR2 (Cell Signaling, 2479,1:150), GFRA3 (R&D, AF670, 1:300), PAX2 (R&D, AF3364, 1:500), F3 (R&D, AF2339, 1:500), PLVAP ( BioRad, MCA2539GA,1:300), GATA3 (R&D, AF2605, 1:300), PECAM1 (BD Pharmingen, BDB550274, 1:300), EHD3 (Novus Biologicals, NBP2-31896, 1:300), FOXC2 (R&D, AF6989, 1:300).

Techniques: In Situ Hybridization, Recombinant, Multiplex Assay, Expressing, Software

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–C) Western blot analysis of (A) pro- and activated caspase-1 (CASP1; P45 and P20, respectively); pro-, activated, and inactivated gasdermin D (GSDMD; P53, P30, and P20, respectively); and pro- and activated gasdermin E (GSDME; P53 and P34, respectively); (B) pro- and cleaved caspase-8 (CASP8; P55 and P18, respectively); pro- and cleaved caspase-3 (CASP3; P35 and P19/P17, respectively); pro- and cleaved caspase-7 (CASP7; P35 and P20, respectively); and (C) phosphorylated and total MLKL (p-MLKL and t-MLKL) in wild-type (WT) and Nlrc5−/− bone marrow-derived macrophages (BMDMs) at 0 h or following treatment with heme plus Pam3CSK4 (Pam3) for 36 h. For loading control, β-actin is shown. (D and E) Cell death images (D) and quantification of cell death (E) using WT, Nlrc5−/−, Nlrp12−/−, and Nlrc5−/−Nlrp12−/− (DKO) BMDMs in response to heme plus Pam3 treatment for 42 h. (F) Western blot analysis of CASP1, GSDMD, GSDME, CASP8, CASP3, CASP7, p-MLKL, and t-MLKL in WT, Nlrc5−/−, Nlrp12−/−, and DKO BMDMs at 0 h or following treatment with heme plus Pam3 for 36 h. For loading control, β-actin is shown. (G) Measurement of IL-1β release in the supernatant of WT, Nlrc5−/−, Nlrp12−/−, and DKO BMDMs following treatment with heme plus Pam3 for 36 h or 42 h. Scale bar = 50 μm (D). Nlrc5−/− BMDMs (ref. 56) (A–G) 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 (E and G). Statistical analyses were performed using the one-way ANOVA (E) or the two-way ANOVA (G). ns, not significant; **P < 0.01; ****P < 0.0001. See also Figure S4.

Article Snippet: After blocking non-specific binding with 5% skim milk, membranes were incubated overnight with the following primary antibodies against: Myc (#2276, CST, 1:1000), NLRP3 (#AG-20B-001, AdipoGen, 1:1000), FLAG (#F1804, Sigma, 1:1000), HA (#2367, CST, 1:1000), human caspase-8 (#ALX-804-242, Enzo Life Science, 1:1000), and GAPDH-HRP (#166574, Santa Cruz, 1:5000).

Techniques: Western Blot, Derivative Assay, Control

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: After blocking non-specific binding with 5% skim milk, membranes were incubated overnight with the following primary antibodies against: Myc (#2276, CST, 1:1000), NLRP3 (#AG-20B-001, AdipoGen, 1:1000), FLAG (#F1804, Sigma, 1:1000), HA (#2367, CST, 1:1000), human caspase-8 (#ALX-804-242, Enzo Life Science, 1:1000), and GAPDH-HRP (#166574, Santa Cruz, 1:5000).

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: (A) Western blot analysis of NLRC5 expression in wild-type (WT), Tlr2−/−, Tlr4−/−, and Tlr2−/−/Tlr4−/− (Tlr2−/−/4−/−) bone marrow-derived macrophages (BMDMs) treated with heme plus Pam3CSK4 (Pam3) for the indicated times. (B) Images representing cell death occurrences in WT, Nlrc5−/−, Tlr2−/−, Tlr4−/−, and Tlr2−/−/4−/− BMDMs at 0 h or following treatment with heme plus Pam3 for 42 h. (C and D) Images representing cell death occurrences (C) and quantification of cell death (D) in WT BMDMs at 0 h or following treatment with heme plus Pam3 with and without nicotinamide (NAM) for 42 h. (E) Western blot analysis of pro- and activated caspase-1 (CASP1; P45 and P20, respectively); pro-, activated, and inactivated gasdermin D (GSDMD; P53, P30, and P20, respectively); pro- and activated gasdermin E (GSDME; P53 and P34, respectively); pro- and cleaved caspase-8 (CASP8; P55 and P18, respectively); pro- and cleaved caspase-3 (CASP3; P35 and P19/P17, respectively); pro- and cleaved caspase-7 (CASP7; P35 and P20, respectively); phosphorylated and total MLKL (p-MLKL and t-MLKL) in WT BMDMs following treatment with heme plus Pam3 with and without NAM for the indicated times. For loading control, β-actin is shown. (F–H) Western blot analysis of NLRC5 in WT BMDMs stimulated with heme (F), Pam3 (G), or heme plus Pam3 (H) with and without NAM for the indicated times. For loading control, α-Tubulin is shown. (I) Quantification of total fluorescence intensity of cellular ROS in WT and Nlrc5−/− BMDMs in response to media alone or heme plus Pam3 treatment with and without NAC or NAM for 36 h. Scale bar = 50 μm (B and C). Nlrc5−/− BMDMs (ref. 56) (B and I) 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 and I). Statistical analyses were performed using the one-way ANOVA (D and I). ns, not significant; ****P < 0.0001. See also Figure S5.

Article Snippet: After blocking non-specific binding with 5% skim milk, membranes were incubated overnight with the following primary antibodies against: Myc (#2276, CST, 1:1000), NLRP3 (#AG-20B-001, AdipoGen, 1:1000), FLAG (#F1804, Sigma, 1:1000), HA (#2367, CST, 1:1000), human caspase-8 (#ALX-804-242, Enzo Life Science, 1:1000), and GAPDH-HRP (#166574, Santa Cruz, 1:5000).

Techniques: Western Blot, Expressing, Derivative Assay, Control, Fluorescence

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: After blocking non-specific binding with 5% skim milk, membranes were incubated overnight with the following primary antibodies against: Myc (#2276, CST, 1:1000), NLRP3 (#AG-20B-001, AdipoGen, 1:1000), FLAG (#F1804, Sigma, 1:1000), HA (#2367, CST, 1:1000), human caspase-8 (#ALX-804-242, Enzo Life Science, 1:1000), and GAPDH-HRP (#166574, Santa Cruz, 1:5000).

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, Expressing, Software