Figure 6.

Phenotypic analysis of dominant-negative INCENP mutant in mammalian cells. (A) INCENP_1–405 inhibits colony growth. Colonies of 16–32 HeLa cells were microinjected with plasmid constructs encoding either INCENP_1–405 or INCENP_43–839 at time t = 0 (abscissa). The aggregate number of cells in all injected colonies is summed on the ordinate. In this experiment, 52% of the cells in colonies injected with INCENP_1–405 were binucleate. This contrasts with 8% binucleate cells in colonies injected with INCENP_43–839 and 4% binucleate cells in uninjected colonies. (B) Expression of INCENP_1–405 causes the accumulation of cells in prometaphase and in ana/ telophase. Slides were prepared from cultures transfected either with the pECE vector alone (158 cells scored), or the vector encoding either INCENP_1–405 (129 cells scored) or INCENP_43–405 (75 cells scored). For each transfected construct, mitotic cells were scored blind for their morphology in mitosis. For the latter two transfections, only cells expressing the transfected INCENP were included in the scoring. (C) For a more detailed analysis of prometaphase congression, a separate series of transfections was performed. Slides were prepared from cultures transfected either with the pECE vector alone (87 cells scored), or the pECE vector encoding either INCENP_1–405 (100 cells scored) or INCENP_43–405 (96 cells scored). As in B, transfected cells were scored blind for their morphology in mitosis.