Fig 2: Identification and characterization of chimeric ECs in normal and tumor-bearing liver tissues. a Representative IHC image of the pan-EC marker CD31 (green), the Kupffer cell marker Clec4f (red) and the nuclear marker DAPI (blue). Top: Compressed z-stack (deconvoluted) of confocal image, arrow head indicates double-positive cell, arrow indicates single-positive vascular structure; Bottom: single optical plane with orthogonal view of single channels and overlay of double-positive cell. b Combined clustering analysis on Kupffer cells and ECs revealed two distinct clusters (7 and 8) between ECs and Kupffer cells. c Top, gene and UMI counts per cluster (b); Bottom, percent contribution of cells to cluster 7 and 8 by sample type and mouse strain, respectively, with exact cell counts next to the plot in the same order as in the stacked bar plot. d Violin plots showing that cluster 7 and 8 express Kupffer/macrophage and EC marker genes as well as myeloid genes at various levels. The numbers on x-axis match to the cluster numbers in the t-SNE plot (b). e Trajectory analysis of all myeloid, Kupffer-EC and macrophage-EC subpopulations. Kupffer-EC (red) and macrophage-EC (pink) cells are highlighted in dashed boxes

Fig 3: In hepatic F4/80+ cells, immune-related genes and pathways are upregulated in SLD-fed Dpp4–/– mice but unchanged in HFHC-fed Dpp4–/– and Dpp4hep–/– mice.Pathway scores of immunological pathways in F4/80+ cells isolated from liver tissue of (A) SLD-fed Dpp4+/+ (n = 6) and Dpp4–/– (n = 6) mice, (B) HFHC-fed Dpp4+/+ (n = 5) and Dpp4–/– (n = 4) mice, and (C) HFHC-fed Dpp4GFP (n = 3) and Dpp4hep–/– (n = 7) mice. (D) Log2-normalized mRNA count of Dpp4 in F4/80+ cells isolated from liver. Liver mRNA abundance (relative to Actb) of (E) Adgre1 and (F) Ccr2. Liver mRNA abundance (relative to Actb) of (G) Itgax, (H) Mrc1, (I) Cd163, (J) Arg1, and (K) Clec4f. (L) Log2-normalized mRNA counts of senescence-associated signaling phenotype (SASP) genes in F4/80+ cells. Log2-normalized mRNA counts of genes associated with (M) NF-?B signaling pathway in F4/80+ cells in SLD-fed Dpp4+/+ (n = 7) and Dpp4–/– (n = 5) mice, HFHC-fed Dpp4+/+ (n = 11) and Dpp4–/– (n = 5–6) mice, and HFHC-fed Dpp4GFP (n = 4) and Dpp4hep–/– (n = 11) mice. Box-and-whisker plots: box extends from the 25th to 75th percentiles; the whiskers go down to the smallest value and up to the largest. Data are presented as the means ± SEM, analyzed by unpaired Student’s t test with Welch’s correction; *P = 0.01–0.05, **P = 0.001–0.01, ***P = 0.0001–0.001, and ****P < 0.0001.

Fig 4: CLEC4F is specifically expressed by Kupffer cells in the mouse liver.A The sequencing results of Clec4f-/- mice with deletion of 4-bp “GCGG” in both Clec4f alleles. B Clec4f mRNA expression in the mouse liver analyzed by qRT-PCR. C CLEC4F protein levels of mouse liver lysates analyzed by western blot. D CLEC4F protein levels of different mouse tissue lysates analyzed by western blot. E Representative images of immunofluorescent staining of formalin-fixed paraffin-embedded mouse liver sections. Kupffer cell (CLEC4F, red or F4/80, green). Results are representative of at least three experiments.

Fig 5: Kupffer cell CLEC4F is a dominant receptor in mediating the rapid clearance of desialylated platelets in the liver.A Flow cytometry analysis of galactose exposure (RCA I binding) on platelets of either WT or Clec4f-/- recipients treated with Arthrobacter ureafaciens-derived neuraminidase at different time points. MFI, Mean fluorescence intensity. B Percentage of platelets of either WT or Clec4f-/- recipients treated with Arthrobacter ureafaciens-derived neuraminidase at different time points. Platelet count was measured using flow cytometric analysis of peripheral blood based on anti-CD41. C Representative immunofluorescent images showing association of platelets (CD41, red) with Kupffer cells (F4/80, blue) in the liver sections. Endothelium, (CD31, green). D, E Quantification of association of platelets with liver Kupffer cells or Kupffer cell (KC) number at 2 h after a neuraminidase injection. n = 5–10 images/10 × fields/experiment. Data represent means ± SEM from three experiments. *P < 0.05.