Figure 6: Effects of β-TRCP-mediated degradation of Set8 on cell cycle progression.

(a) U2OS cells harbouring Flag-Set8^WT, Set8^ΔPIP, Set8^S253A or Set8^ΔPIP/S253A were cultured in the presence of 0.1 μg ml⁻¹ doxycycline (Dox). Cell cycle was analysed by propidium iodide staining using fluorescence-activated cell sorting. Data are shown as mean±s.d. of three independent experiments. *P<0.05, Student’s t-test. (b) U2OS cells harbouring inducible Flag-tagged Set8^WT, Set8^ΔPIP, Set8^S253A or Set8^ΔPIP/S253A were synchronized in S phase with hydroxyurea (HU) for 24 h. During the last 4 h in HU, expression of Set8 was induced by Dox. Then, cells were released from HU in the presence of Dox, and IB analysis was performed to detect the indicated proteins. (c) U2OS cells harbouring inducible Flag-tagged Set8^WT, Set8^ΔPIP, Set8^S253A or Set8^ΔPIP/S253A were synchronized in M phase with nocodazole for 18 h. During the last 4 h in nocodazole, expression of Set8 was induced by Dox. Then, cells were released from nocodazole in the presence of Dox, and IB analysis was performed to detect the indicated proteins. (d) A schematic illustration of the signalling network that timely regulates the Set8 protein stability during the cell cycle progression.