FIG.7.

Brd4 and SPA-1 regulate G₂/M progression. (A) Scheme of cell synchronization and transfection. HeLa cells were transfected in G₂, 8 h after release from double thymidine block. Cells were harvested 10 h after transfection, stained with propidium iodide, and analyzed by flow cytometry. (B) Cell cycle profiles of cells transfected with indicated plasmids (3 μg). (C) The percentages of cells in different phases of the cell cycle are shown for indicated transfections. Values represent the averages of three independent experiments. (D) Immunoblotting of total extracts from the harvested cells to detect ectopic Brd4, Brd4 mutants, and SPA-1 expression. As a loading control the same filters were immunoblotted with antibodies to α-tubulin. (E) HeLa cells were synchronized by double thymidine block and transfected with Brd4-expressing vectors in G₁, 12 h after release from double thymidine block. Cells were harvested 8 h after transfection, stained with propidium iodide, and analyzed by flow cytometry. The percentages of G₁, S, and G₂/M populations are shown for indicated transfections on the left. Values represent the averages of three independent experiments. Immunoblot analyses to verify overexpression of Brd4 and SPA-1 are shown on the right.