Fig. 3.

O-GlcNAcylation promotes colorectal cancer metastasis by regulating the EMT via EZH2. a Morphological changes by O-GlcNAcylation in SW620 and SW480 cells. Photographs using the × 20 objective. b–d Western blotting and immunofluorescence staining reveal an increased localization of epithelial markers in the membrane and downregulated expression of mesenchymal markers in SW620-Sh-1 and SW620-Sh-2 cells. In contrast, the upregulation of O-GlcNAcylation after 12 h of treatment with PUGNAc (100 mol/L) or Thiamet-G (10 μmol/L) resulted in a decreased localization of epithelial markers in the membrane and increased expression of mesenchymal markers in SW480 cells. e Workflow used for the identification of putative O-GlcNAc-modified proteins in SW480 cells. f A representative list of putative O-GlcNAc-modified proteins. The protein IDs from UniProt and gene symbols are shown. g The MS/MS spectrum and sequencing results of an unmodified peptide from in-gel digestion. The peptide was identified as AIQTGEEIFFDYR, corresponding to the 715–727 fragment of EZH2. h, i Western blot showing the levels of EZH2 protein and H3K27me3 in SW480 cells transfected with ShRNA-OGT (Sh-2 and Sh-3) or the control vector (h) or SW480 cells after a 12-hour treatment with PUGNAc (100 mol/L), Thiamet-G (TMG, 10 μmol/L), or isometric DMSO (negative control, NC) (i). The mean level of the indicated protein from three independent biological replicates is shown on the right. The values shown are expressed as the means ± SEM. ** represents Student’s t test P < 0.01 and ***P < 0.001. j, k Transwell assay and western blot analysis of the indicated proteins in SW480 cells after a 12-hour treatment with TMG (10 μmol/L) alone or in combination with GSK-343 (3 μmol/L) for 3 days. Isometric DMSO treatment was used as a negative control. l, m Transwell assay and western blot analysis of the indicated proteins in SW480 Lv-OGT cells transfected with EZH2 siRNA or negative control