FIGURE 6.

FBXO31 depletion induces re-replication. HeLa cells were transiently transfected with control, FBXO31, Skp2 or FBXO31 (siRNA-2), and Skp2 siRNAs 72 h before harvest. Cells were also transfected with empty vectors, Cdt1 and vector, or Cdt1 and FBXO31 expression plasmids. The transfected cells were synchronized by a double-thymidine block and harvested at the indicated time points after release from the block. All the samples were pulsed with EdU for 30 min before harvest. The samples were analyzed by flow cytometry. A, dot plots of the 7-AAD area versus 7-AAD width were used to gate for single cells only as shown for HeLa cells transfected with control si at time 0. B, the EdU profile and cell cycle analysis data are presented as dot plots. The arrowheads indicate cells with 2C, 4C, 6C, and 8C DNA content. The EdU and cell cycle profile (7-AAD) of cells at 9 h are also presented as histograms in the right panels. C, bar graph showing the relative percentage of EdU positive cells. Western blotting was carried out with anti-FBXO31, anti-Skp2, anti-Cdt1 and anti-γ-tubulin antibodies.