Fig 2: FUT4 was directly targeted by miR-320c. (a) The target relationship between miR-320c and FUT4 was predicted using starBase software. (b and c) The relationship between miR-320c and FUT4 was confirmed via dual-luciferase reporter and RIP assays. (d and e) Expression of FUT4 in C28/I2 cells transfected with miR-320 mimic or its inhibitor was assessed via qRT-PCR and Western blot assays. (f) TUG1 level in OA was analyzed by qRT-PCR. (g–j) The mRNA and protein expression of FUT4 in separated chondrocytes and IL-1ß-stimulated C28/I2 cells were examined by qRT-PCR and Western blot assays, respectively. (k) Spearman’s correlation analysis was used to analyze the interrelation between miR-320c and TUG1. *P < 0.05.

Fig 3: Schematic diagram shows the mechanism that TAMs promote Ezrin phosphorylation mediated EMT in lung adenocarcinoma through FUT4/LeY -mediated fucosylation

Fig 4: FUT4/LeY-mediated fucosylation of Ezrin was closely associated with the phosphorylation of Ezrin(A) A549 and H1299 cells were treated with TCM in different concentration (0%–25% or 50%) for 48 h. The expression of p-Ezrin (T567), N-cadherin, Vimentin and Snail were analyzed by western blot analysis. (B) A549/H1299 cells and A549-shFUT4/H1299-shFUT4 cells were treated with indicated dose of TCM for 48 h. The expression of Ezrin, p-Ezrin (T567), N-Cadherin, Fibronectin, Vimentin, ZEB1 and Snail were analyzed by western blot analysis. (C) A549 and H1299 cells preincubated with tunicamycin for 4 h. Then cells were stimulated with indicated dose of TCM for 24 h. The expression of p-Ezrin (T567), N-cadherin, Vimentin and snail were analyzed by western blot analysis. (D) Schematic diagram of the potential N-glycosylation sites on the Ezrin protein. (E) Cell lysates were immunoprecipitated with either IgG or Ezrin antibody and detected by western blot analysis as indicated. (F) Cell lysates were immunoprecipitated using Ezrin antibody, and the samples were separated using SDS-PAGE followed by blotting biotinylated UEA lectin or Ezrin antibody. (G) A549 cells and A549-shFUT4 cells were transfected with WT Ezrin plasmids or vector. Then cells were stimulated with indicated dose of TCM for 48 h. The expression of p-Ezrin (T567), N-cadherin, Vimentin and Fibronectin were analyzed by western blot analysis. (H) A549 cells and A549-shFUT4 cells were transfected with T567D-mutant Ezrin plasmids or vector. Then cells were stimulated with indicated dose of TCM for 48 h. The expression of p-Ezrin (T567), N-cadherin, Vimentin and Fibronectin were analyzed by western blot analysis. Every experiment was conducted at least three times, and the average is shown (mean ± SD). *P < 0.05, **P < 0.01, significant.

Fig 5: FUT4 overexpression reversed the effects of miR-320c on cell proliferation, apoptosis and degradation in IL-1ß-treated C28/I2 cells. (a–f) C28/I2 cells underwent IL-1ß stimulation were transfected with miR-NC, miR-320c, miR-320c + pcNDA or miR-320c + FUT4, respectively. (a and b) The mRNA and protein levels of FUT4 were evaluated by qRT-PCR and Western blot assays, respectively. (c) Cell proliferation was measured using MTT assay. (d) Apoptotic cells were calculated via flow cytometry. (e and f) Western blot assay was carried out to detect the protein expression of cyclin D1, cleaved-casp-3, MMP-13, collage II and Aggrecan. *P < 0.05.