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. 2017 Nov 14;8:1491. doi: 10.1038/s41467-017-01654-6

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 6 — O-GlcNAcylation of SIRT1 protects cells from death during stress stimuli. a Flag-tagged wtSIRT1 or SIRT1^S549A was co-expressed with or without p53 in NCI-H1299 cells, and then the cells were treated with 2 μM MG132, 1 μM TSA and either 25 μM etoposide or solvent for 2 h. The whole-cell extracts were immunoblotted with antibodies against p53 acetylated at Lys 382, p53, SIRT1 or β-tubulin. Data represent two independent experiments. b NCI-H1299 cells, which were stably silenced endogenous SIRT1 and reintroduced exogenous wtSIRT1 or SIRT1^S549A, were transfected with/without p53 and treated with etoposide. The percentage of cell apoptosis/death were analyzed by Muse Annexin V & Dead Cell Kit on Muse Cell Analyzer. Data represent three independent experiments. c NCI-H1299 cells, which were stably silenced endogenous SIRT1 and expressed wtSIRT1 or SIRT1^S549A, were treated with 2 μM MG132, 1 μM TSA and either 25 μM etoposide or solvent for 2 h. FOXO3 proteins were immunoprecipitated and detected by IB with antibodies against acetylation, FOXO3, SIRT1 or β-tubulin. Data represent two independent experiments. d NCI-H1299 cells, which were pretreated with or without 25 μM etoposide for 1 h, were lysed and labeled with GalNAz and biotin. The biotinylated proteins were enriched by STV-conjugated magnetic beads, and then the total protein samples (input), the immunoprecipitated protein samples (IP) and the remaining supernatant samples (S) were probed with indicated antibodies. Data represent two independent experiments. e Schematic representation of a model of how SIRT1 O-GlcNAcylation at S549 regulates its deacetylase activity and cellular response to stress stimuli. Student’s t-test. Results are expressed as the mean ± s.d. (n = 3 biologic replicates). P-values: ns (not significant), ***P < 0.001