Figure 4.

Failure in microtubule nucleation in PKD3 deficient MEFs: Wild type and PKD3 deficient MEFs were kept under normal cell culture conditions before fixation and incubation with indicated antibodies according to protocols described in material and methods. (A) α/β-tubulin antibody staining of wt (first three rows on the left) and PKD3^−/− (last three rows on the right) MEFs at indicated mitotic phases (estimated by DAPI staining). (B) γ-tubulin antibody staining of wt (first three images on the left) and PKD3^−/− (last three images on the right) MEFs at metaphase (estimated by DAPI staining). (C) First row: Wild type MEFs were treated with 50μM H₂O₂ for 15 min at 37°C before fixation and indicated staining or antibody incubation according to protocols described in material and methods. This sample served as control for the validation of cytoplasmic Cyt C release after apoptotic induction. Second row: Example of a PKD3 deficient MEF with appropriate aligned condensed chromosomes at prometaphase. Third row: Example of a PKD3 deficient MEF with improper aligned condensed chromosomes at prometaphase. Applied stainings and antibody are indicated above the images. Cyt C release into the cytoplasm (green color) indicates the induction of the apoptotic cascade. All images are representatives of many for the indicated staining. Scale bars: 48 μM.