Figure 1. PLK1T210D/+ cells enter mitosis with normal kinetics and have an intact G2 DNA-damage checkpoint.
a Schematic of PLK1 locus and gene-targeting vectors. Triangles represent FRT sites. b–c HCT116 PLK1+/+ and PLK1T210D/+ cells were synchronized by a double thymidine block and released into medium containing nocodazole. At 90-minute intervals, samples were withdrawn for immunoblotting of the indicated proteins (b) and determination of mitotic indices by Hoechst staining and fluorescence microscopy (c). Error bars indicate SEM. d G2 checkpoint assay (Papi et al. 2005). HCT116 PLK1+/+ and PLK1T210D/+ cells were pre-treated with caffeine (caf) to induce checkpoint override or left alone, challenged with adriamycin (adr) or ionizing radiation (IR) as indicated, and then cultured in the presence of nocodazole for 6 hours to trap cells escaping from G2 phase into mitosis. Mitotic indices were determined as above. e hTERT-RPE1 PLK1+/+ and PLK1T210D/+ cells were treated with nocodazole (Noc) or nocodazole plus the Aurora A kinase inhibitor MLN8054 (Noc + MLN) for 4 hr. Mitotic indices were then determined and compared to untreated cells.