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. 2021 Jul 9;11:14253. doi: 10.1038/s41598-021-93684-w

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© The Author(s) 2021

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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SIRT6 deacetylates CDH1, a co-activator of APC/C, and promotes its degradation. (a) Control and SIRT6-FLAG overexpressing 293 T and HeLa cells were subjected to western blotting with the indicated antibodies. HSP70 was used as a loading control. (b) Level of CDH1 protein was detected by western blotting in wild-type and SIRT6 knockout 293 T and HeLa cells. GAPDH and HSP70 were used as loading controls. (c) Control (EV) and SIRT6-overexpressing (ST6) HeLa cells were synchronized to G1, S, G2 and M phases, respectively. Levels of CDH1, FLAG, cyclin B1 and phosphorylation of histone H3 at Ser10 (pH3@Ser10) were detected with corresponding antibodies. GAPDH was used as loading control. (d) Control (EV) and SIRT6-overexpressing (ST6) cells were treated with 5 μg/mL cycloheximide (CHX) for 0, 2, 4, and 8 h. Levels of CDH1 were detected by western blotting. Actin was used as loading control. (e) Tandem mass spectrometry spectrum of the acetylated peptide for identification of Lys135 modifications in CDH1. An asterisk in the peptide sequence indicates the modified lysine residue. The image was generated with Xcalibur 3.0 (Thermo Fisher Scientific Inc.). (f) Synthesized peptide around CDH1 K135 with acetylation was incubated with SIRT6 without/with NAD⁺. Samples were analyzed with MALDI-TOF/TOF with m/z range 1400–1600 (z = 1). (g) 293 T stably expressing wild-type or K135Q CDH1-HA were transfected with empty vehicle (EV) or SIRT6-FLAG expressing vector (ST6), respectively. CDH1-HA levels were detected using anti-HA antibody. Expression of SIRT6-FLAG was detected using an anti-FLAG antibody. Actin was used as a loading control.