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. Author manuscript; available in PMC: 2021 Sep 1.
Published in final edited form as: Biochemistry. 2019 Nov 18;59(34):3169–3179. doi: 10.1021/acs.biochem.9b00560

Figure 4.

Figure 4.

GFAT inhibition sensitizes cancer cells to undergo apoptosis under oxidative stress. (A) GFAT can be inhibited by the natural product 6-diazo-5-oxo-norleucine (DON). (B) Oxidative stress induces a small increase in O-GlcNAcylation that is inhibited by DON treatment. A549, H1299, or MCF7 cells were treated with diamide to induce oxidative stress and/or DON to inhibit GFAT before analysis by Western blotting. (C) DON and diamide cotreatment results in increased apoptosis. A549, H1299, or MCF7 cells were treated with the indicated concentrations of diamide and/or DON, and caspase-3/7 activity was measured using the Caspase-Glo 3/7 Assay (Promega) (n = 3). (D) Exogenous GlcNAc rescues cells from apoptosis. A549, H1299, or MCF7 cells were treated with the indicated concentrations of diamide, DON, and/or GlcNAc, and caspase-3/7 activity was measured using the Caspase-Glo 3/7 Assay (Promega) (n = 3). (E) Exogenous GlcNAc rescues O-GlcNAc levels during DON treatment. A549, H1299, or MCF7 cells were treated with DON and/or GlcNAc, and O-GlcNAcylation levels were visualized using Western blotting. Data shown in panels C and D represent the mean ± s.e.m. Statistical significance was determined using a one-way ANOVA test followed by the Dunnett test.