FIG. 1.

Expression of kinase-inactive Nek6 and Nek7 mutants induces apoptosis. (A) HEK 293 cells were transiently transfected with FLAG-tagged Nek6 and Nek7 constructs, as indicated, for 24 h before cells were lysed and subjected to immunoprecipitation with anti-Flag antibodies. The amount of kinase precipitated was determined by Western blotting with anti-Flag antibodies and the immunoprecipitates used for kinase assays, with β-casein as a substrate. Autoradiographs (³²P) are shown of the Nek6/Nek7 and β-casein proteins. WT, wild type. (B) Activity of the different Nek6 and Nek7 constructs is expressed as a percentage of wild-type activity normalized to the amount of precipitated protein. The data are shown as means ± standard deviations from three separate experiments. (C) HeLa cells were transiently transfected with Flag-tagged Nek6 and Nek7 constructs for 24 and 48 h before being assayed for their ability to take up annexin V/FITC stain via flow cytometry analysis. As a positive control, untransfected cells were treated with 1 μM staurosporine (ST) for 4 h. (D) HeLa cells were transiently transfected with Flag-tagged Nek6 and Nek7 constructs for 48 h before being fixed and stained with Flag antibodies to detect transfected cells and cleaved caspase 3 antibodies to detect apoptotic cells. Data in panels C and D represent means (±standard deviations) from counts of at least 50 cells in three separate experiments. (E) RPE1-hTERT cells were transiently transfected with FLAG-tagged Nek6-WT and Nek6-K75M constructs for 48 h before being fixed and stained with anti-FLAG and anti-cleaved caspase 3 antibodies. Data represent means (±standard deviations) from counts of at least 100 cells in three separate experiments. (F) HeLa cells were transiently transfected with FLAG-tagged Nek6-WT and Nek6-K75M constructs for 48 h with and without treatment with 500 ng/ml nocodazole before being fixed and stained with anti-FLAG antibodies to detect transfected cells and anti-cleaved caspase 3 antibodies to detect apoptotic cells. Data represent means (±standard deviations) from counts of at least 100 cells in three separate experiments. (G) HeLa cells were transiently transfected with Flag-Nek6 constructs for 48 h in the presence or absence of 1 mM hydroxyurea before being assayed for their ability to take up annexin V/FITC via flow cytometry analysis. Data represent means (±standard deviations) from three separate experiments. In all experiments, untransfected cells (−) were used as a negative control.