Figure 1.

SET8 negatively controls the level of UHRF1 protein and is responsible for UHRF1 downregulation in G2/M. (A) UHRF1 is highly expressed in the S phase of cell cycle in human HFL1 fibroblast cells. HFL1 cells were cultured with addition of 5-ethyl-2'-deoxyuridine (EdU) for 2 h, followed by detection of EdU by click chemistry and UHRF1 by immunostaining. In a representative experiment, 65 out of 68 counted UHRF1 highly expressed cells were cells in S phase. (B) Western blot analysis of the levels of SET8, UHRF1 and DNMT1 proteins in different stages of cell cycle in four different cell lines. (C and D) Western blot and RT-qPCR analyses showing that knockdown of SET8 increased the level of UHRF1 proteins and had no effect on the level of UHRF1 mRNA in HFL1 cells (C) and HeLa cells (D). (E) Immunostaining showing that knockdown of SET8 by siRNA increased the number of non-S phase cells with highly expression of UHRF1. In a representative experiment, in the control 95 out of 100 UHRF1-highly expressed cells were in S phase, whereas only 75 out of 100 were in S phase with SET8 knockdown. (F) Western blot analysis showing that knockdown of SET8 by two different SET8-specific shRNAs impaired UHRF1 downregulation in G2/M phase of cell cycle in HFL1 and HeLa cells. (G) IP-western analysis showing an interaction between ectopically expressed UHRF1 and SET8.