Figure 3
p27K induces mitotic catastrophe and centrosome reduplication. (a) p27WT, p27C, p27K and p27CK cells received Dox for 72 h or were left untreated (UT). Percent annexin-positive cells was determined by flow cytometry. (b) Top panels: p27WT, p27C, p27K and p27CK cells received Dox for 60 h. Cells were immunostained with antibodies to p27Kip1 (red) and α-tubulin (green). Middle and bottom panels: Parental (Par), p27WT, p27C, p27K and p27CK cells received Dox for 30 h. Cells were immunostained with antibodies to p27Kip1 (red) and γ-tubulin (green). Nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole) (blue). Micrographs were taken with a Leica TCS SP5 AOBS laser scanning confocal microscope (Leica Microsystems, Germany) through a 100 × /1.40 NA Plan Apochromat oil immersion objective lens. Areas in the boxes are enlarged below. White arrows denote micronuclei. (c) Centrosome numbers per cell were determined. Middle panels of (b) and data in (c) are from the same experiment. At least 200 cells per sample were examined with an Axio Imager Z1, Carl Zeiss upright fluorescent microscope at 63 × /1.40 NA oil objective lens. Binucleated cells were not counted.
