File S1 - CLEC4F Is an Inducible C-Type Lectin in F4/80-Positive Cells and Is Involved in Alpha-Galactosylceramide Presentation in Liver

Figure S1, S2, S3, S4, S5 and Table S1. Figure S1 in File S1. Generation of Clec4f knockout mice. (A) Clec4f targeting strategy. Genomic structure of the wild-type and targeted alleles of Clec4f gene was destroyed by inserting the EGFP gene into the exon 4 of Clec4f gene. (B) Southern blot hybridization of genomic DNA from wild-type (+/+), heterozygous (+/−) and homozygous (−/−) offspring by using 3′-flanking probe. (C) Western blot analysis of CLEC4F protein expression from liver extracts. B: BamHI; E: EcoRI; EV: EcoRV; H: HindIII. Figure S2 in File S1. Characterization of CLEC4F expression by murine tissue array screening. Immunohistochemistry of CLEC4F (B, D, F, H, J, L, N, P, R) in mouse tissues, including liver (A, B), BM (C, D), thymus (E,F), spleen (G, H), LN (I, J), brain (K, L), lung (M, N), kidney (O, P) and gut (G, R) and mIgG1 was used as isotype control (A, C, E, G, I, K, M, O, Q). Sections were counterstained with hematoxylin. BM, bone marrow; LN, lymph node; M, medulla; C, cortex; W, while pulp; R, red pulp; F, follicle. Scale bar: 100 µm. Figure S3 in File S1. CLEC4F expression screening. (A) CLEC4F-transfected 293T cells, (B) macrophage cell lines, RAW264.7 and J774, and Kupffer cell line, KC13-2, and (C) hepatic cell populations were used for CLEC4F staining. Cells were stained with lineage markers: F4/80, NK1.1, B220 or TCR, and double or triple stained with CLEC4F. Mouse IgG1 was used as isotype control. Signals were determined by FACS Calibur for flow cytometric analysis. Figure S4 in File S1. The expression of CLEC4F is decreased under culture condition. Kupffer cells isolated from C57BL/6 mice were cultured in RPMI 1640 supplemented with 10% (v/v) heat-inactivated fetal bovine serum at 37°C in 5% CO2. The expression of CLEC4F was detected by Western blot in indicated culture time periods. Figure S5 in File S1. CLEC4F is dispensable for C. albicans infection in vivo. (A) The survival rate of wild-type and Clec4f−/− littermates after C. albicans infection. The p value was determined by using Kaplan-Meier survival analysis and compared by Log-rank test. (B) Kupffer cells (1×105 cells) isolated from wild-type and Clec4f−/− littermates were stimulated with heat-killed conidia (HK C), heat-killed hyphae (HK H) and live conidida (L C) in 1×106 CFU/ml. The supernatant were collected at 24 h post-stimulation and detected the level of IL-6 and TNF-α by ELISA. *: p (PDF)