Extended Data Fig. 8: CHK1 promotes WEE1 phosphorylation at Ser642.
From: SIRT1 deacetylates WEE1 and sensitizes cancer cells to WEE1 inhibition
(a) U2OS cells were irradiated with 40 J/m2 UV and allowed to recover for the indicated time. (b) U2OS cells were pre-treated with CHK1 inhibitor MK-8776 (2 μM) or ATR inhibitor VE-821 (10 μM) for 1 hr prior to UV irradiation. (c) A U2OS cell line to express SFB-WEE1 under the control of a tetracycline-inducible promoter was generated. The resulting cells transfected with the indicated siRNAs were treated with doxycycline 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. (d) A MDA-MB-231 cell line to express 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 (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. P-values were determined by two-way ANOVA. (e) MDA-MB-231 cells were either synchronized in G1/S phase by a double thymidine (Thd) block or in mitosis using nocodazole (Noc) treatment. Cells were then released into the cell cycle and collected at the indicated time points. (f) MDA-MB-231 cells were synchronized in G1/S phase. Cells were then released into the cell cycle and collected at the indicated time points. (g) MDA-MB-231 cells were synchronized in G1/S phase. Cells were then treated with CHK1 inhibitor MK-8776 (2 μM), released into the cell cycle, and collected at the indicated time points. (h) MDA-MB-231 cells were either synchronized in G1/S phase or in mitosis. Cells were then released into the cell cycle and collected at the indicated time points. The co-immunoprecipitated GCN5/SIRT1 levels were normalized with immunoprecipitated WEE1 levels expressed as the normalized ratio (shown below the blots). Data in A-B, E-H were independently replicated at least three times, with similar results.
