PUGNAc is a well-investigated inhibitor for protein-O-GIcNAcase, whereas recent investigations showed that PUGNAc had a broad range as inhibitor for cellular beta-hexosaminidases. Here we report that PUGNAc treatment provokes globotetraosylceramide (Gb4Cer) accumulation in human umbilical vein endothelial cells (HUVEC). HPLC analysis and a quantitative ELISA using newly developed anti-Gb4Cer monoclonal antibody revealed that PUGNAc treatment specifically increased the expression of Gb4Cer among glycosphingolipids expressed in HUVEC. Although the effect was weaker than PUGNAc, an O-GIcNAcase selective inhibitor (Thiamet-G) treatment also increased Gb4Cer levels in HUVEC. Furthermore, both of PUGNAc and Thiamet-G treatment up-regulated the expression levels of alpha-1,4-galactosyltransferase/ Gb3Cer synthase gene which encodes a key enzyme in Gb4Cer synthesis. These results indicate that protein-O-GlcNAcylation can regulate the expression levels of cellular Gb4Cer. (C) 2017 Elsevier Inc. All rights reserved.