Fig. 5. O-GlcNAcylation of c-Rel is required for TCR-induced, CD28RE-dependent gene expression.

(A and B) PUGNAc increases anti-CD3– and anti-CD28–induced, c-Rel–dependent gene expression in Jurkat cells. Cells were treated with 100 µM PUGNAc for 12 hours and then were treated with plate-bound anti-CD3 and anti-CD28 antibodies (each at 2 µg/ml) for 3 hours. Samples were then analyzed by quantitative real-time polymerase chain reaction (PCR) to determine the abundances of (A) IL2 and (B) CSF2 mRNAs relative to that of transferrin receptor (TFRC) mRNA. **P < 0.01, ***P < 0.001, when comparing PUGNAc-treated to untreated samples. (C to G) Jurkat T-REx cell clones were incubated with doxycycline for 22 hours to induce production of WT c-Rel or S350A mutant c-Rel, left untreated or treated with PUGNAc overnight, and then were left untreated or were treated with anti-CD3 and anti-CD28 antibodies for 3 hours. Samples were then analyzed by quantitative real-time PCR to determine the abundances of (C) IL2, (D) CSF2, (E) IFNG, (F) NFKBIA, and (G) TNFAIP3 mRNAs relative to that of TFRC mRNA. **P < 0.01, ***P < 0.001, when comparing cells expressing WT and mutant c-Rel under similar treatments. ns, not significant. (H) Jurkat T-REx cell clones expressing WT c-Rel or mutant c-Rel were treated overnight with 2 mM STZ and then were treated as described in (C). ***P < 0.001, when comparing STZ-treated samples to untreated samples. Data in (A) to (H) are means ± SEM of triplicate samples and are representative of three independent experiments, with similar results.