Figure 8.

Overexpression of Aur-A can override the Golgi checkpoint in HeLa cells. HeLa cells (A–C) were arrested in S phase by using the double-thymidine block and transfected for 24 h with the empty vector (GFP; C only), GFP-Aur-A, or GFP-Plk1 (C only) after the first release from thymidine. (A) Representative images of cells labeled with an antibody against giantin (red) to label the Golgi complex, with DRAQ5 (blue) for the cell cycle phase. (B) Representative images of cells labeled with an antibody against phospho-T288-Aur-A (red) and with DRAQ5 (blue) for the cell cycle phase. (C) One hour after the final thymidine washout, the cells were either nonmicroinjected (noninj) or microinjected with recombinant GRASP65 (8–10 mg/ml), and with TRITC-conjugated dextran as microinjection marker. The cells were fixed 12 h after the final thymidine washout, and processed for immunofluorescence under confocal microscopy, with antibodies against GFP to identify low transfection levels, and with Hoechst 33342 for cell cycle phase. Quantification of the relative mitotic index as percentages of microinjected cells in mitosis were normalized to nonmicroinjected cells on the same coverslip. More than 400 cells were microinjected for each condition. All quantification data are means ± SD from three independent experiments, each carried out in duplicate. Bar, 5 μm.