FIGURE 5.

Depletion of MCM10 induces G₂/M arrest and complementation of wild-type MCM10 inhibits Vpr-induced G₂/M arrest. A, HEK293T cells were double-transfected with siRNA against MCM10 or non-targeting siRNA (NT) with 24-h intervals. Forty-eight hours after the second transfection, cells were analyzed for depletion of MCM10 and the cell cycle was analyzed by propidium iodide staining. B, complementation of MCM10 inhibits Vpr-induced G₂/M arrest. HEK293T cells were co-transfected with increasing amounts of MCM10-HA expressor and a constant amount of FLAG-Vpr expressor. The total amount of transfected DNA was kept constant by adding empty vector. Cell cycle was analyzed after 48 h. C, complementation of ΔCTD MCM10 does not inhibit Vpr-induced G₂/M arrest. HEK293T cells were co-transfected with increasing amounts of ΔCTD MCM10-HA expressor and a constant amount of FLAG-Vpr. After 48 h, the cell cycle was analyzed. D, ΔCTD MCM10 does not bind Vpr and VprBP. HEK293T cells were co-transfected with plasmids for expression of FLAG-Vpr and the full-length HA-tagged MCM10 (WT) or CTD truncated MCM10 (ΔCTD). After 48 h, cells were lysed and immunoprecipitated with anti-HA antibody conjugated to agarose beads. All experiments were repeated at least two times, and one representative experiment is shown.