Fig. 6. Aurora A kinase activity is required for spindle bipolarity. Transgenic lines encoding RNAi-resistant Aurora A transgenes were transfected with siRNAs designed to repress Aurora A or Lamin B1, then exposed to tetracycline as indicated, to induce transgene expression. (A) Immunoblot showing simultaneous repression of endogenous Aurora A and induction of Myc-tagged Aurora A D-N. The arrow indicates the Myc-tagged exogenous Aurora A, whereas the asterisk indicates endogenous protein. (B) Immunofluorescence images showing monopolar spindles in Aurora A RNAi cells expressing the Aurora A transgenes. In panels i and iii, the horizontal arrows indicate bipolar Aurora-A-positive spindles in untransfected cells and the arrowheads indicate prometaphase-like Aurora-A-deficient cells. In panels ii and iv, the vertical arrows indicate bipolar or monopolar spindles, respectively, in cells expressing the Aurora A transgene. Bars, 10 µm. (C) Bar graph quantifying monopolar spindles showing that although the wild-type Aurora A rescues the RNAi phenotype, the Aurora A D-N kinase mutant does not. The values represent the mean ± s.e.m. derived from three independent experiments in which at least 100 mitotic cells were scored.