FIGURE 7.

De novo SET8 reaccumulation after S phase is necessary for normal mitotic progression. A, illustration of the experimental approach to block SET8 reaccumulation only after S phase. B, HCT116 cells were synchronized in early S phase via double thymidine block. Cells were released into fresh medium containing control (luciferase) or SET8-ORF siRNA and harvested at the indicated time points for immunoblotting of the indicated endogenous proteins (*, a nonspecific background band). C, cells were synchronized as in A and then released into siRNA-containing medium for 10 h prior to fixation and immunofluorescence staining for tubulin and DAPI staining for DNA. Scale bar, 5 μm. D, the number of mitotic cells from C in preanaphase versus anaphase/telophase was quantified in three biological replicates (n = 300 for each replicate) using two different siRNAs as indicated. Error bars, S.E.