Generation of a human KIF4A knockout cell line. (A) Cloning strategy for the generation of a recombinant adeno-associated virus (rAAV) gene targeting construct. (B) HCT116 WT and KIF4A KO clone F1 were analyzed by PCR (top), immunoblotting (middle), and immunofluorescence (bottom), demonstrating correct targeting and the resulting loss of KIF4A protein. (C) HCT116 wild-type and KIF4A KO cells were immunostained for α-tubulin (green), kinetochores (red), pericentrin (pink), and DNA (blue) and confocal z-stacks of anaphase and telophase cells acquired. Deconvolved MIPs are shown. Arrows indicate disorganized midzone MTs. Bar, 5 µm. (D) HCT116 wild-type and five different KIF4A KO clones were seeded at 0.5 × 105 cells per well and cell numbers determined over 7 d. Graphs represent the mean of four independent experiments (±SEM). (E) HCT116 wild-type and KIF4A KO cells were monitored by phase-contrast video live-cell microscopy during a period of 16 h. Mitosis was slightly prolonged in HCT116 KIF4A KO cells as shown in the box-and-whisker plot. (F) Cells from E were classified dependent on their fate during and after mitosis, respectively.
