Amino acid functional domains of CD248 protein in human, mouse and zebrafish. Red squares, signal peptide; Blue rectangles, transmembrane region; CLECT, C-type lectin domain; EGF, epidermal growth factor-like domain; EGF_CA, Calcium-binding EGF-like domain; CCP, complement control protein domain.

Colinearity analysis between zebrafish cd248a, cd248b and human CD248. All genes were indicated by arrows, and the arrow direction represented the gene direction. Silver strip, chromosome. Chr, chromosome.

Phylogenetic trees of Cd248a and Cd248b. Phylogenetic trees constructed from the amino acid sequences of homologous genes of various species. CD248, CD93 and CLEC14A are members of the C-type lectin group 14 family. (A) neighbor-joining method; (B) maximum-likelihood method. The reliability of each node was estimated by bootstrapping with 1000 replications. The numbers shown at each node indicate the bootstrap values (%).

WISH of cd248a in zebrafish early embryos. Stages of embryonic development: (A) 2-cell; (B) 32-cell; (C) 512-cell; (D) sphere; (E) shield; (F) bud; (G, H) 10 somites; (I) 24 hpf; (J) 48 hpf; (K) 72hpf; (L) 96 hpf; (M) 120 hpf. hpf, hours post-fertilization. Scale bar, 100 μm.

WISH of cd248b in zebrafish early embryos. Stages of embryonic development: (A) 2-cell; (B) 32-cell; (C) 512-cell; (D) sphere; (E) shield; (F) bud; (G, H) 10 somites; (I) 24 hpf; (J) 48 hpf; (K) 72hpf; (L) 96 hpf; (M) 120 hpf. hpf, hours post-fertilization. Scale bar, 100 μm.

Quantitative analysis of zebrafish cd248a and cd248b genes in response to LPS/PBS treatment. (A) Quantitative analysis of zebrafish cd248a in infected embryos/larvae by microinjecting LPS at 1-cell stage; (B) Quantitative analysis of zebrafish cd248b in infected embryos/larvae by microinjecting LPS at 1-cell stage. PBS, Phosphate Buffered Saline; LPS, lipopolysaccharide. Data were shown as mean ± SD, n = 3. *P < 0.05; ***P < 0.001; ****P < 0.0001.

Subcellular localization of Cd248a, Cd248b and truncated Cd248b in HEK293T cells. The pcDNA3.1/V5/Cd248a/eGFP, pcDNA3.1/V5/Cd248b/eGFP and pcDNA3.1/V5/eGFP recombinant plasmids were transfected individually into HET293T cells. The nucleus was stained by DAPI. One representative image for each out of three independent experiments is shown. Scale bar, 50 μm.

Effect of overexpression of zebrafish cd248a and cd248b on pro-inflammatory cytokines expression in mouse macrophages. The pcDNA3.1/V5/Cd248a, pcDNA3.1/V5/Cd248b and pcDNA3.1/V5-His A recombinant plasmids were transfected individually into RAW264.7 cells. At 24h after transfection, the total RNA of the cells was extracted and cDNA was synthesized. Quantitative analysis of the mRNA expression of mouse Tnfα, Il1β and Il6. Data were shown as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. ns, not significant.

Effect of overexpression of zebrafish cd248a and cd248b on pro-inflammatory cytokines expression in vivo. Diluted mRNAs were injected into one-cell stage embryos of zebrafish. At 48h after injection, the total RNA of the embryos was extracted and cDNA was synthesized. (A, C) Quantitative analysis of the expression of tnfα, il1β and il6 after cd248a and cd248b overexpression in embryos; (B) Quantitative analysis of the expression of hif1a, pigf and vegfr1 after cd248a overexpression in embryos; (D) Quantitative analysis of the expression of notch3 and mmp9 after cd248b overexpression in embryos. cd248a-OE, cd248a overexpression; cd248b-OE, cd248b overexpression. Data were shown as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Effect of cd248 deletion on inflammatory cytokines. The mutants of cd248a and cd248b were carried out by CRISPR/Cas9 system. (A–C) CRISPR/Cas9 gRNA design and the genotyping result of the cd248a-mutant allele; (D–F) CRISPR/Cas9 gRNA design and the genotyping result of the cd248b-mutant allele; (G, H) Quantitative analysis of the expression of cd248a or cd248b after cd248a-deletion or cd248b-deletion compared with WT respectively; (I) Quantitative analysis of the expression of zebrafish tnfα, il1β and il6 in the cd248-mutants and LPS treatment. (J) Quantitative analysis of the expression of il4 in the cd248-mutants compared with WT. Data were shown as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. ns, not significant.