TGF- treatment was found to enhance GalNAc-T enzyme activity for the synthetic peptide substrate derived from the IIICS domain defining the FDC6 epitope. 1 M EtDO-P4, the specific inhibitor of GlcCer synthase (32), did not cause a significant switch in cell morphology, manifestation of EMT marker molecules, or cell motility (Fig. 1 and Fig. S2), though the majority of GSLs were essentially depleted under this condition, as assessed by orcinol/sulfuric acid staining after HPTLC separation of extracted GSLs (Fig. S3and and and and 0.05; ** 0.005; *** 0.001. Our earlier studies with glycopeptides comprising the VTHPGY sequence indicated that mAb FDC6 binding was managed by treatment with sialidase followed by -galactosidase; however, the binding activity was completely lost by endo–lectin, which binds terminal GalNAc residues (Fig. S6). Recently, we found that HUH-7 cells transfected with cDNA for GalNAc-T6 create more FDC6-positive FN compared with the parent cells (Fig. S7). Collectively, these results indicate the down-regulation of onfFN, recognized with FDC6 ROR agonist-1 in the cells transfected with the siRNAs, is definitely accomplished through selective knockdown of the GalNAc-Ts. The effect of the reduction of FDC6-positive FN induced from the siRNAs, on EMT process, was assessed by switch of cell morphology, manifestation of EMT marker molecules, and cell motility. Transfection of the targeted siRNAs inhibited the switch of cell morphology induced by TGF- treatment in the both cell lines, whereas transfection of the control siRNA experienced no significant effect (Fig. 4). As expected, the expression of total FN, defined by EP5, was not significantly affected by the knockdown, and was enhanced to similar degree by TGF- treatment in both cell lines, whereas TGF-Cinduced up-regulation of onfFN, defined by FDC6, was strongly inhibited in T6/T3 knockdown cells. In addition to FN in cell lysates, FN secreted in culture supernatants was analyzed, and similar results were obtained. In the knockdown cells, compared with the nontransfected controls and the unfavorable siRNA-transfected controls, expression of the mesenchymal markers Ncad and vimentin was significantly lower, and the expression of the epithelial cell marker Ecad was higher in both WPE (Fig. 5) and PNT1a cells (Fig. S8). The enhanced cell motility induced by TGF- treatment was strongly inhibited in the knockdown cells, but not in the nontransfected control cells or the unfavorable ROR agonist-1 control siRNA transfected cells for the both cell lines (Fig. 6). Open in a separate windows Fig. 4. Effect of knockdown of GalNAc-T3/T6 on TGF-Cinduced EMT, assessed by cell morphology. WPE and PNT1a cells were transfected with a mixture of siRNA duplexes for human GalNAc-T3 and T6 to obtain double knockdown cells, or with unfavorable siRNA for control cells, and then treated with TGF- as explained in 0.05; ** 0.005. ROR agonist-1 (and and 0.001. Conversation The EMT process was originally observed through in vivo studies of cells in tissues associated with early embryonic development (36). The process was later found to play a key role in tissue repair, to prevent apoptosis and senescence, and to induce characteristic properties of stem cells. EMT is also reported to be a cause of organ fibrosis and to promote malignancy progression through enhancement of cell motility, acquisition of stem cell characteristics, and other mechanisms (10C15). In view of the many documented examples of aberrant glycosylation associated with malignancy progression (37C40), we assumed that glycosylation changes in GSLs or glycoproteins occur during the EMT process. Our previous studies using the mouse mammary epithelial cell collection NMuMG demonstrated Mouse monoclonal antibody to eEF2. This gene encodes a member of the GTP-binding translation elongation factor family. Thisprotein is an essential factor for protein synthesis. It promotes the GTP-dependent translocationof the nascent protein chain from the A-site to the P-site of the ribosome. This protein iscompletely inactivated by EF-2 kinase phosporylation a functional role of Gg4: Expression of Gg4 was reduced by down-regulation of Gg4 synthase gene expression during EMT, and enhancement of Gg4 level inhibited EMT (16, 17). In contrast, the present study using EtDO-P4, the inhibitor of GlcCer synthase (32), did not show involvement of GSLs in EMT process in prostate epithelial cell lines WPE and PNT1a. However, we cannot rule out the possible involvement of GSL having GalCer as core structure; for example, Gal1C4Gal1-Cer (diGalCer) (41), Gal-sphingosine (psychosine), two types of plasmalopsychosine (42), and NeuAc2C3Gal1-Cer (GM4 ganglioside) ROR agonist-1 (43) have.