Fig. 6: FUT2 promotes fatty acid synthesis in CRC cells via fucosylation of YAP1.

A Co-immunoprecipitation assay to determine the interaction between FUT2 and YAP1/TAZ in SW480 cells, indicating direct protein-protein interactions of FUT2 with YAP1. B SW480 cells were transfected with FUT2-shRNA or treated with 1 μM 2F-PAF for 24 h. Following the treatment, western blot analysis of YAP1 expressions were assessed after UEA-1 pull-down. C Visualization of the glycosylation sites on YAP1 and SREBP-1, showing the potential sites of modification. D SW480 cells were transfected with FUT2-shRNA. After transfection, the cells were treated with 1 μM 2F-PAF for 24 h, and then western blot analysis of CTGF and p-YAP1 expressions were determined. The relative density of protein bands was quantified, normalized to β-actin, and fold changes were shown in histograms from three independent experiments. E SW480 cells were transfected with FUT2-shRNA and YAP1-siRNA. After transfection, cells were analyzed for western blot analysis of indicated proteins. The relative density of protein bands was quantified, normalized to β-actin, and fold changes were shown in histograms from three independent experiments. F SW480 cells were transfected with YAP1-siRNA, and then the mRNA levels of YAP1, FASN, and CPT1A were assessed by qRT-PCR. Data shown are mean ± SD; n = 3 samples. G, H SW480 cells were transfected with FUT2/Lv105. After transfection, cells were treated with verteporfin (500 nM) for 24 h, and then and then stained with Nile red (G) or phalloidin (H) to evaluate intracellular cytoskeleton organization. Green or red fluorescence signals were captured and visualized through a fluorescent microscope using constant fluorescence parameters explained in the methods section: scale bar, 50 μm. I Design of the tumor-establishment studies. BALB/c nude mice were subcutaneously implanted with the indicated FUT2-knockdown (sh-FUT2 SW480) or control SW480 (shRNAcont) cells. J After 30 days of transplantation, western blot analysis of ACC1, FASN, YAP1, and TAZ expression in isolated tumors (n = 6 mice/group) from the SW480 xenograft model were determined. The relative density of protein bands was quantified, normalized to β-actin, and fold changes were shown in histograms from three independent experiments. Statistical significance was determined using one-way ANOVA using the Bonferroni post-hoc test, with *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: not significant.