Fig 2: O-GlcNAcylation of PGK1 is important for colon cancer cell proliferation.a Analysis of PGK1 glycosylation in a panel of three human colon cancer cell lines and a normal colon cell line NCM460. b Cell proliferation of PGK1 WT or T255V rescue cells. In RKO, LoVo and HT-29 cells, endogenous PGK1 was depleted with shRNA targeting PGK1, and then rescued with ectopic expression of PGK1 WT or PGK1 T255V, which is resistant to shRNA targeting. c Cell proliferation of PGK1 WT or T255V rescue HT-29 cells in the presence of various 3-PG and ATP concentrations. d Cell cycle analysis of PGK1 WT or T255V rescue RKO, LoVo and HT-29 cells. Error bars denote the means ± standard deviations (SD). Statistical analyses were performed by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001). Source data are provided as a Source Date file.

Fig 3: T255 O-GlcNAcylation of PGK1 promotes tumor formation and increases in colon tumor tissues.a Tumor formation in nude mice injected with PGK1 WT or T255V rescue HT-29 cells. Dissected tumors at the experimental endpoint were shown, and the masses of the dissected tumors were weighted. b Immunoblotting analysis of PGK1 glycosylation and OGT expression in human colon tumor (T) tissues and the matching adjacent tissues (N). c–e The statistical analysis of paired tumor and matching adjacent tissues. Relative PGK1 glycosylation level was normalized to the total PGK1 protein level for each patient. Source data are provided as a Source Date file.

Fig 4: T255 O-GlcNAcylation activates PGK1 enzyme activity.a Enzymatic activity of PGK1 under basal conditions, upon OGT overexpression or with TMG treatment. b Enzymatic activity of PGK1 upon cellular treatment with different concentrations of H2O2 or diamide. c Comparison of enzymatic activity of PGK1 WT and different mutants upon OGT overexpression. Error bars denote the means ± standard deviations (SD). Statistical analyses were performed by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001). Source data are provided as a Source Date file.

Fig 5: T255 O-GlcNAcylation of PGK1 regulates glucose metabolism in cells.a Comparison of glucose uptake, lactate production, ATP levels, and the total energy charge (ADP/ATP ratio) in PGK1 WT or T255V rescue HT-29 cells in the presence or absence of OGT overexpression. b The extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) in PGK1 WT or T255V rescue HT-29 cells in the presence of OGT overexpression. c–f Relative abundance of metabolites derived from glycolysis (c), TCA cycle (d), the pentose phosphate pathway (e), and the serine synthetic pathway (f) in PGK1 WT or T255V rescue HT-29 cells in the presence of OGT overexpression. Error bars denote the means ± standard deviations (SD). Statistical analyses were performed by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001). Source data are provided as a Source Date file.