FIG 3.
Vpr+ RTECs undergo aberrant mitosis following a prolonged G2 phase. (A) (Left) Schematic indicating how DNA content (measured by propidium iodide [PI] staining) and mitosis (measured by anti-phospho-histone H3 [pH3]) can distinguish between the G0/G1, G2, and M phases. (Center) Flow cytometry plot at 0 min after thymidine release. The percentage of each discernible cell cycle stage is given. (Right) Flow chart showing the steps performed for thymidine synchronization of HK2 cells. (B) Time course of DNA content or percentage of mitotic cells over time in synchronized HK2 control cells and HK2 cells transduced with HR-HA-VprΔGFP. (Left) Graphical representation of flow cytometry analysis of the percentage of cells with 4C DNA content at each time point. (Right) Percentage of mitotic (pH3+) cells within that 4C population at each time point. (C) Immunofluorescence microscopy of mitosis in synchronized HK2 cells 18 h after transduction with HR-HA-VprΔGFP. A total of 22.1% of mitotic Vpr+ cells, had spindle defects, including multipolar spindles (Vpr1, Vpr2), in contrast to only 6.0% of control cells (627 control cells and 461 Vpr cells [P, <0.00001 by the chi-square test]).