Fig. 5.

Stress stimuli induced O-GlcNAcylation and activation of SIRT1. a Stress stimuli induced SIRT1 O-GlcNAcylation in a time-dependent manner in NCI-H1299 cells (top). NCI-H1299 cells were stimulated with etoposide (25 μM), H₂O₂ (200 μM) or glucose depletion (fasting) for the indicated times and O-GlcNAcylation of SIRT1 was detected by chemoenzymatic labeling and IB analysis. Data represent two independent experiments. b, c NCI-H1299 cells were transfected with Flag-tagged wtSIRT1 or SIRT1^S549A, followed by treatment with etoposide (25 μM for 1 h), H₂O₂ (200 μM for 30 min) or glucose depletion (1 h). SIRT1 proteins were immunoprecipitated, and their O-GlcNAcylation levels (b) and deacetylase activities (c) were detected by chemoenzymatic labeling and fluorometric assay, respectively. Data represent two independent experiments. d–f Balb/c mice were either fasted for 18 h or injected intraperitoneally with 200 μl of 100 mM H₂O₂. SIRT1 proteins were immunoprecipitated from whole liver extracts. Chemoenzymatic labeling and IB analysis were used for the detection of SIRT1 O-GlcNAcylation levels (d). Mouse livers were homogenized in NP40 lysis buffer supplied with or without HexNAcase inhibitors, and SIRT1 proteins were immunoprecipitated from whole liver extracts. SIRT1 O-GlcNAcylation levels (e) and deacetylase activity (f) were detected by chemoenzymatic labeling and fluorometric assay system, respectively. For animal experiments, six mice per group were used, representative data from two independent experiments. For chemoenzymatic labeling, samples from two mice were mixed in equal amounts for detection. Student’s t-test. Results are expressed as the mean ± s.d. (c, n = 3 biologic replicates; f, n = 6 mice per group). P-values: ns (not significant), *P < 0.05, **P < 0.01, ***P < 0.001