Fig 2: Influences of catechins (CAs: EC, epicatechin; ECG, epicatechin-3-gallate; EGC, epigallocatechin; EGCG, epigallocatechin-3-gallate) on TLR4/CD14 pathway in LPS-triggered RAW264.7 cells. The expression levels of CD14, TLR4, MD-2, MyD88, and TRAF6 in cells. Results were displayed as mean ± SD from no less than three experimental replications. Diverse lowercases in the histogram denote significant differences (p < 0.05).

Fig 3: Expression pattern of acute phase response genes is distinct in AI and GI glands. Quantitative RT-PCR analysis of A. Stat3, B. C/EBPδ, C. CtsL, D. CtsB, E. Orm1, F. Orm2, G. Lrg1, H. Slpi, I. CD14, J. Chi3L1, and K. MMP2. Expression normalized to RPL37. n = 3 per group per time point. Li. Western blot analysis and quantitation of Lii. pStat3Y705, Liii. Stat3, Liv. CtsB, and Lv. CD14. M. Temporal pattern of pSTAT3Y705 positive cell distribution in AI and GI glands between day 7 and day 28 postpartum. n = 6 per group per time point. Ni. Representative images of FFPE sections of AI (day 11 PPM) and GI (day 22 and 25 PPM) mammary glands subjected to immunofluorescence analysis of pStat3Y705 (green) and CD14 (red). DAPI (blue) was used to counterstain the nuclei. Nii. Bar diagram showing the number of pStat3.Y705 and CD14 double-positive cells per field of view (FOV) in mammary gland sections of AI (day 11 PPM) and GI (day 22 and 25 PPM). n = 5 per group per time point. Number of FOV = 10

Fig 4: TRIF determines inflammatory output of Fas through RIP1K activation(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.

Fig 5: CD14 is required for the internalization of the Fas receptor in macrophages(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.