FIG. 2.

Kinase-inactive Nek6 and Nek7 trigger mitotic delay at two stages. (A to C) HeLa cells were either untransfected or transfected, as indicated, with Flag-tagged Nek6-wild type (WT), Nek6-K75M, or Nek6-S206A for 48 h before being fixed and stained with antibodies against Flag (green in merge) to detect transfected cells and cleaved caspase 3 (red in merge) to detect apoptotic cells. DNA was stained with Hoechst 33258 (blue in merge). Cells in interphase (A), metaphase (B), and cytokinesis (C) are shown. Scale bar, 10 μm. (D) HeLa cells were either untransfected (−) or transiently transfected with different Flag-Nek6 and Flag-Nek7 constructs, as indicated, for 24 h before being processed for immunofluorescence microscopy with Flag antibodies to detect transfected cells and α-tubulin antibodies to detect the mitotic spindle. The histogram represents the observed frequency of transfected cells in the different stages of mitosis indicated. Data are means (±standard deviations) from three separate experiments, in which at least 50 cells were counted for each construct.