Extended Data Fig. 6: Acetylation is required for UV-induced CDK1 Tyr15 phosphorylation.
From: SIRT1 deacetylates WEE1 and sensitizes cancer cells to WEE1 inhibition
(a) WEE1 acetylation is required for G2/M checkpoint activation following DNA damage. A U2OS cell line to express siRNA-resistant SFB-WEE1 under the control of a tetracycline-inducible promoter was generated. The resulting cells were transfected with the indicated siRNAs and then treated with doxycycline (1 μg/mL) to induce the expression of WEE1. 8 hr later, cells were irradiated with 10 J/m2 UV and allowed to recover for 4 hr. Percentages of mitotic cells were determined by FACS analysis. Results shown are means of three independent experiments and were presented as means ± SEM. P-values were determined by two-tailed Student’s t-test. (b, c) Knockdown of GCN5 sensitizes cells to MK-1775. Results shown are means of three independent experiments and were presented as means ± SEM. P-values were determined by two-way ANOVA. (d, e) SIRT1 depletion delays mitotic entry and induces cell apoptosis. Percentages of mitotic and apoptotic cells were determined by FACS analysis. Results shown are means of three independent experiments and were presented as means ± SEM. P-values were determined by two-tailed Student’s t-test. (f) HEK293T cells were transfected with the indicated plasmids for 24 hr. Cell lysates were then prepared and western blot analysis was carried out as indicated. (g) SFB-WEE1 immunopurified from HEK293T cells were incubated with SFB-CDK1 in a kinase reaction mixture in the presence or absence of MK-1775 (10 μM). (h, i) SIRT1 sensitizes cells to MK-1775 by deacetylating WEE1. A MDA-MB-231 cell line to express siRNA-resistant SFB-WEE1 under the control of a tetracycline-inducible promoter was generated. The resulting cells were infected with lentiviruses expressing control or SIRT1 shRNAs and then transfected with the indicated siRNAs. 36 hr post-transfection, cells were treated with doxycycline (1 μg/mL for 24 hr) and indicated doses of MK-1775 for 48 hr. Results shown are means of three independent experiments and were presented as means ± SEM (I). P-values were determined by two-way ANOVA. Knockdown efficiency was confirmed by western blotting (H). Data in F-G were independently replicated at least three times, with similar results.
