Journal: Cell reports

Article Title: CD14 is a decision-maker between Fas-mediated death and inflammation

doi: 10.1016/j.celrep.2024.114685

Figure Lengend Snippet: (A) Representative 40× images of TRIF or RIPK1 staining and colocalization in permeabilized macrophages of indicated genotypes stimulated with mFasL for 1 h. (B and C) Quantification of total TRIF puncta (B) and TRIF puncta colocalized with RIP1K puncta (C). Each data point represents a FOV across 3 technical replicates, 5 FOVs per replicate, presented with mean ± SD. (D) FADD-specific immunoprecipitation in B6 or Cd14 −/− macrophages stimulated with mFasL for indicated timepoints and probed for death complex components. (E–G) Cell death over time in primary murine neutrophils, peritoneal macrophages, and BMDMs isolated from indicated genotypes as measured by propidium iodide incorporation over time. (H) Level of cleaved, active form of death caspases-8, -3, and -7 over time in B6 and Cd14 −/− macrophages after FasL stimulation. See also . Data from cell death assays and western blotting are representative of three or more independent experiments, and cell death data are presented as the means ± SD of triplicate wells All blots from a specific image are run from a single set of lysates that were identically handled. Data from imaging experiments are representative of three or more independent experiments, and data points indicate FOVs, error bars indicate ± SD, n = 15 (~20 cells/FOV). ANOVA was used for comparison between groups: ns ( p > 0.05), ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: CD14 (mouse) , Cell Signaling Technology , Cat#93882; RRID:AB_2800216.

Techniques: Staining, Immunoprecipitation, Isolation, Western Blot, Imaging, Comparison

Journal: Cell reports

Article Title: CD14 is a decision-maker between Fas-mediated death and inflammation

doi: 10.1016/j.celrep.2024.114685

Figure Lengend Snippet: (A) Serum levels of LDH 2–4 h post-mFasL tail vein injection of 0.3 μg/mL. (B and C) TUNEL staining (B) and quantification of TUNEL+ cells per μm2 (C) in spleens 4 h post-tail vein injection of 0.3 μg/mL. Each point represents the four averaged FOVs of spleen section from an individual mouse, presented with mean ± SD (B6 n = 3, Cd14 −/− n = 3, PBS data points represent data from B6 n = 3 and Cd14 −/− n = 1). (D) Serum and spleen levels of CXCL1, TNF, and IL-10 2–4 h post-mFasL injection of 0.3 μg/mL or PBS control. See also and . Each point represents an individual mouse, presented with mean ± SD. ANOVA was used for comparison between groups: ns ( p > 0.05), ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: CD14 (mouse) , Cell Signaling Technology , Cat#93882; RRID:AB_2800216.

Techniques: Injection, TUNEL Assay, Staining, Control, Comparison

Journal: Cell reports

Article Title: CD14 is a decision-maker between Fas-mediated death and inflammation

doi: 10.1016/j.celrep.2024.114685

Figure Lengend Snippet: (A) Levels of CXCL1 produced by macrophages of indicated genotype in response to FasL over time. Data points indicate individual mice as source of isolated macrophages. (B) IκBα and total and phosphorylated p65 over time in whole-cell lysates from B6, Cd14 −/− , and Trif −/− macrophages after FasL stimulation. (C, E, and F) Relative IFNB and ISG15 mRNA levels normalized to ACTB in response to FasL over time in indicated neutrophils and macrophages. (D) Total and phosphorylated forms of RIPK1 over time in whole-cell lysates from B6, Cd14 −/− , and Trif −/− macrophages after FasL stimulation. (G) Spleen levels of IFN-β 4 h post-mFasL injection of 0.3 μg/mL; each point represents an individual mouse. qPCR and ELISA data are presented as the means ± SD for duplicate wells from three or more independent experiments. Data from western blotting are representative of three or more independent experiments. ANOVA was used for comparison between groups: ns ( p > 0.05), * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: CD14 (mouse) , Cell Signaling Technology , Cat#93882; RRID:AB_2800216.

Techniques: Produced, Isolation, Injection, Enzyme-linked Immunosorbent Assay, Western Blot, Comparison

Journal: Cell reports

Article Title: CD14 is a decision-maker between Fas-mediated death and inflammation

doi: 10.1016/j.celrep.2024.114685

Figure Lengend Snippet: (A) Cell death of BMDMs transduced with indicated constructs measured over time by propidium iodide incorporation. (B) Representative 40× images of CD14 or FasR staining and colocalization in permeabilized B6 macrophages stimulated with mFasL for 30 min. (C) Quantification of CD14 and Fas puncta. Each data point represents a FOV across 3 technical replicates, ~10 FOV per replicate. (D) Representative 40× images of extracellular FasR staining of non-permeabilized B6 and Cd14 −/− macrophages after 1 h of mFasL stimulation. (E) Quantification of extracellular FasR after 1 h of mFasL stimulation in B6 and Cd14 −/− macrophages. Each data point represents a FOV across 3 experimental replicates, with 3 technical replicates per experiment. (F) Cell death of PMA-differentiated U937-derived macrophages transduced with indicated constructs measured over time by propidium iodide incorporation. (G) Relative Tnf mRNA levels normalized to ACTB in response to FasL in indicated U937-derived macrophages. See also Figure S6. (H) Proposed model depicting role of CD14 in Fas-mediated death with and without pathogens present. Data from cell death assays and qPCR are representative of three or more independent experiments, and cell death data are presented as the means ± SD of triplicate wells (~5,000 cells per FOV). Data from imaging experiments are representative of three or more independent experiments, and data points indicate FOVs, n = 15 (~20 cells/FOV). Image quantification is presented with mean ± SD. ANOVA was used for comparison between groups: ns ( p > 0.05); ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: CD14 (mouse) , Cell Signaling Technology , Cat#93882; RRID:AB_2800216.

Techniques: Transduction, Construct, Staining, Derivative Assay, Imaging, Comparison

Journal: Cell reports

Article Title: CD14 is a decision-maker between Fas-mediated death and inflammation

doi: 10.1016/j.celrep.2024.114685

Figure Lengend Snippet:

Article Snippet: CD14 (mouse) , Cell Signaling Technology , Cat#93882; RRID:AB_2800216.

Techniques: Recombinant, Reverse Transcription, CyQUANT Assay, LDH Cytotoxicity Assay, In Situ, TUNEL Assay, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Software