FIG. 5.

Brd4-dependent recruitment of P-TEFb to mitotic chromosomes. (A) Wild-type (WT) but not mutant Cdk9 S175A becomes associated with mitotic chromosomes before nuclear envelope/lamina reassembly. Stable HeLa-based cell lines expressing Flag-tagged wild-type Cdk9 or Cdk9 mutant S175A were immunostained at the indicated cell cycle stages (right) with antibodies against Flag (red) and lamin B1 (green). Chromosomes in the same cells were stained with DAPI (blue). Bar, 5 μm. (B) siRNA-mediated depletion of Brd4 in HeLa cells. The levels of Brd4 and Cdk9 in cells containing either an empty vector (−) or the Brd4-specific siRNA (+) were analyzed by Western blotting. (C) Reduced association of CycT1 with mitotic chromosomes in Brd4 knockdown cells. HeLa cells with (+) or without (−) the expressed Brd4 siRNA were immunostained with antibodies against CycT1 and α-tubulin to examine the behaviors of CycT1 in mitotic cells. Quantification of the percentages of cells with CycT1 on chromosomes is shown against either total cell population (left) or only mitotic cells (right). Error bar represents the mean ± standard deviation. (D) Association of CycT1 with mitotic chromosomes in cells with incomplete Brd4 depletion. Coimmunofluorescence staining was performed with anti-Brd4 (red) and anti-α-tubulin (green) or anti-CycT1 (green) antibodies in Brd4 knockdown cells. Chromosomes were stained with DAPI (blue). Arrowheads indicate cells with detectable levels of Brd4 that were in mitosis. Bar, 10 μm.