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. 2021 May 11;12:2672. doi: 10.1038/s41467-021-22618-x

Fig. 5. FUT8 is involved in the core fucosylation process of B7H3.

Fig. 5

a The heatmap of 13 fucosyltransferases (FUTs) mRNA expression in breast cancer. The RNA-Seq gene count data of 113 pairs of breast cancer samples and matched adjacent normal samples were retrieved from Gene Expression Omnibus (GSE62944), and further normalized using DESeq2 variance-stabilizing transformation. The heatmap was plotted with relative expression values, which were calculated as fold change to the average expression level in adjacent normal breast tissues. b The correlation between B7H3 and FUT8 at protein levels. Mass spectrometry-based proteomics data for TCGA samples were measured by The Clinical Proteomic Tumor Analysis Consortium (CPTAC), and were downloaded from CPTAC data portal (https://proteomics.cancer.gov/data-portal). All patients were stratified according to PAM50 subtypes as indicated. The relationship was assessed using Pearson’s chi-square test. c LCA affinity of whole-cell lysate of the indicated MDA-MB-231-B7H3KO cell lines by western blot with anti-B7H3. Black closed circle, non-specific band. d Lectin blotting of B7H3 for detecting fucosylation status. Fucosylation of B7H3 in the indicated MDA-MB-231-B7H3KO cell lines expressing sgRNAs targeting FUT8 was probed with LCA after exogenous B7H3 was immunoprecipitated. e Left: Representative images of LCA binding (core fucose) and membrane B7H3 in the indicated MDA-MB-231-B7H3KO cell lines measured by FACS. Right: LCA Median Fluorescence Intensity (MFI) and B7H3 MFI were plotted (n = 3 biological independent samples). Error bars represent mean ± SD. The p value was determined by one-way ANOVA with Dunnett’s multiple comparisons test, no adjustments were made for multiple comparisons. NS, not significance. f HEK293T cells were transiently transfected with Flag-tagged B7H3-WT and HA-tagged FUT8, followed by immunoprecipitation with anti-Flag beads and immunoblot analysis with anti-HA and LCA. g HEK293T cells were transiently transfected with Flag-tagged B7H3-WT and HA-tagged FUT8 or its mutants, followed by immunoprecipitation with anti-Flag beads and immunoblot analysis with anti-HA and LCA. Data are representative of three independent experiments. Red closed circle, glycosylated B7H3; blue star, non-glycosylated B7H3.