Fig. 6

Fucosyltransferase 8 (FUT8) knockout impairs transforming growth factor-β1 (TGF) ligand binding and decreases TGF-β downstream signaling in MDA-MB-231 cells. a Establishment of FUT8-knockout (KO) MDA-MB-231 cells by CRISPR-Cas9-mediated genome editing. Two independent KO #1 and #2 clones targeting exon 3 or 6 of FUT8 were established (upper panel). The deletion of FUT8 and loss of core fucosylation were confirmed by western blot (lower-left panel) and LCA binding assay (lower-right panel) in MDA-MB-231 cells. Of note, FUT8 inactivation did not affect the expression of TGF-β RI or RII protein (lower-left panel). b Core fucosylation of TGF-β RI and RII protein were eliminated by FUT8 KO. Recombinant TGF-β RI and RII proteins produced in the control or two FUT8-KO MDA-MB-231 cell lines were probed with biotinylated Lens culinaris lectin (LCA), then detected by streptavidin-conjugated horseradish peroxidase. c Reporter assay with a TGF-β1-responsive luciferase reporter, 3TP-lux, showed significantly reduced TGF-β1-mediated signaling in FUT8-KO cells, which was rescued completely by FUT8 overexpression. d Schematic figure showing the indirect ligand binding assay. HEK-293 T cells were transfected with empty vector or the expression plasmid encoding HIS-tagged TGF-β1 (HIS.TGF-β1) protein. After 2 days, conditioned medium were added to MDA-MB-231 cells endogenously expressing TGF-β receptors. Bound HIS.TGF-β1 protein was quantified by the alkaline phosphatase (AP)-conjugated anti-HIS antibody reacting with its colorimetric substrate p-nitrophenyl phosphate. e FUT8 knockout reduced TGF-β1 ligand binding. Data are mean ± SD. *P < 0.05, **P < 0.01. mOD405, milli-absorbance units at 405 nm