FIGURE 5:

Binucleate primary cells lose G2/M markers. (A) HFF cells were transfected with lentivirus expressing fluorescent geminin and hCdt1 (chromatin licensing and DNA replication factor 1), markers for G2/M and G1, respectively. After ascertaining that the cells were fluorescent, cells were exposed to 10 μM DCB for 24 h and then were recorded for 24 h after drug release. Video images from DeltaVision microscope recordings were quantitated for cells positive for geminin or hCdt1 markers. In the same culture chamber, mononucleate cells were positive for the G2/M geminin marker, while binucleate cells were negative. Both populations were positive for hCdt1. Data are composite from three independent experiments. Right, microscopy images show geminin (green) mononucleate cells and an hCdt1 (orange) positive binucleate cell. Scale bar, 40 μm. (B) Tetraploid REF52 cells lose late cell cycle markers. REF52 and TAG cells were exposed to 10 μM DCB for 24 h, released for 6 h, and then exposed again to DCB for 24 h to maximize REF52 tetraploidy. Western blots of cell extracts taken at the indicated times after final drug release show that REF52 cells have lost Aurora B and cyclin B by 48 h of release, whereas TAG cells continue to express these late-cell-cycle proteins. α-Tubulin serves as a loading control. NT gel lanes were from the same gel as treated samples, but they were not contiguous, as indicated by separations.