Figure 4: O-GlcNAcylation of G6PD promotes cellular biosynthesis, cell proliferation and antioxidant defense.

(a) DNA synthesis in WT and S84V G6PD replacement A549 cells under hypoxic conditions, as determined by 5-bromo-2’-deoxyuridine incorporation assays. Control experiment was performed in G6PD depleted A549 cells (n=4 assays). (b) Lipogenesis in WT and S84V G6PD replacement A549 cells under hypoxic conditions, as determined by ¹⁴C-glucose labelled lipid incorporation assays. Control experiment was performed in G6PD depleted cells (n=3 assays). (c) Cell proliferation rates under hypoxic conditions of WT and S84V G6PD replacement A549 cells. Rescue experiments were carried out by the addition of GSH (5 mM), Nuc (four ribonucleotides and four deoxyribonucleotides), or both in the culture medium. Control experiments were performed in G6PD depleted A549 cells (n=3 assays). Cell numbers were determined by trypan blue counting. (d) NADPH and (e) GSH levels in WT and S84V G6PD replacement A549 cells in the absence or presence of OGT overexpression under normoxic conditions (n=3 assays). (f) Cellular reactive oxygen species (ROS) levels induced by different concentrations of diamide in WT and S84V G6PD replacement A549 cells for 2 h under normoxic conditions (n=4 assays). (g) Percentage of cell death induced by varying hydrogen peroxide concentrations in WT and S84V G6PD replacement A549 cells for 1 h under normoxic conditions (n=4 assays). Error bars denote mean±s.d. of three independent experiments. Statistical analysis was performed by one-way analysis of variance and Bonferroni comparison post-test in c, and Student’s t-test in other experiments (*P<0.05).