O -Linked N -Acetylglucosamine Transiently Elevates in HeLa Cells during Mitosis

-linked -acetylglucosamine ( -GlcNAc) is a dynamic post-translational modification of serine and threonine residues on nuclear and cytoplasmic proteins. -GlcNAc modification influences many cellular mechanisms, including carbohydrate metabolism, signal transduction and protein degradation. Multiple studies also showed that cell cycle might be modulated by -GlcNAc. Although the role of -GlcNAc in the regulation of some cell cycle processes such as mitotic spindle organization or histone phosphorylation is well established, the general behaviour of -GlcNAc regulation during cell cycle is still controversial. In this study, we analysed the dynamic changes of overall -GlcNAc levels in HeLa cells using double thymidine block. -GlcNAc levels in G₁, S, G₂ and M phase were measured. We observed that -GlcNAc levels are significantly increased during mitosis in comparison to the other cell cycle phases. However, this change could only be detected when mitotic cells were enriched by harvesting round shaped cells from the G₂/M fraction of the synchronized cells. Our data verify that -GlcNAc is elevated during mitosis, but also emphasize that -GlcNAc levels can significantly change in a short period of time. Thus, selection and collection of cells at specific cell-cycle checkpoints is a challenging, but necessary requirement for -GlcNAc studies.