Degradation of SET8 allows cell cycle progression beyond G1 phase. (A) Nondegradable SET8 reduces entry into mitosis. U2-OS cells were cotransfected with H2B-GFP and the indicated Flag-HA-SET8 constructs in a 1:3 ratio and simultaneously treated with aphidicolin for 24 h. Cells were released into fresh medium containing nocodazole, harvested, and fixed at the indicated time points. Cells were stained with H3S10P antibody, and cells positive for both GFP and H3S10P were identified by flow cytometry. Data are presented as mean + SD. (B) Expression levels of SET8 constructs. U2-OS cells were treated with nocodazole for 16 h (Noco) or transfected with the indicated pBabe or pCMV constructs 36 h before harvest. Cells were processed for immunoblotting with the indicated antibodies. Only 0.1 of the pCMV SET8 WT lysate was loaded compared with pBabe SET8 lysates. (C) SET8 WT and SET8-*PIP2 expression induces compaction of chromatin. pCMV Flag-HA-SET8 constructs were expressed in U2-OS cells seeded on coverslips and simultaneously treated with aphidicolin for 24 h. Cells were released into fresh medium and fixed after 4 h. Chromatin structure in SET8-expressing cells was analyzed by immunofluorescence stainings for DAPI in HA-positive cells. Bars, 10 µm. (D) Elevated levels of SET8-*PIP2 but not SET8-*PIP2/*SET results in a more compact chromatin structure. HEK293 cells were transfected with either Flag-HA-SET8-*PIP2 or Flag-HA-SET8-*PIP2/*SET constructs. Cells were harvested for MNase digest followed by separation of the DNA on an agarose gel. (E) Elevated levels of SET8-*PIP2 but not SET8-*PIP2/*SET results in a more compact chromatin structure in response to UV. HEK293 cells were transfected with either Flag-HA-SET8-*PIP2 or Flag-HA-SET8-*PIP2/*SET constructs. Cells were treated with UV (100 J/m²) 3 h before harvest. Cells were harvested for MNase digest followed by separation of the DNA on an agarose gel.