Compromised spindle checkpoint function in BUBR1^+/– MEFs. (A) Wild-type and BUBR1^+/– MEF cells were lysed, and an equal amount of cell lysates was blotted for BUBR1 and for α-tubulin. (B) Independent BUBR1 signals from paired MEFs were quantified via densitometry, and average amounts of BUBR1 signals after normalization by α-tubulin signals were shown. (C) BUBR1^+/– and wild-type MEFs were incubated with nocodazole (0.2 μg/mL) for the indicated times, after which cell lysates were subjected to immunoblot analysis with antibodies to cyclin B or to α-tubulin. (D) BUBR1^+/– MEFs stained with antibody to α-tubulin (green) and with DAPI (blue) were examined by fluorescent microscopy. Two representative polyploid cells were shown. Arrows denote micronuclei. Bar = 5 μm. (E) Polyploid cells in both BUBR1^+/– and wild-type MEFs were scored from 500 cells, and the percentage of cells with polyploidy is shown. (F) MEF cells were transfected with siRNA targeting human BUBR1 or luciferase gene mRNA for 3 days, after which equal amounts of cell lysates, along with untransfected parental lysates, were subjected to immunoblot analysis with antibodies to BUBR1 or to α-tubulin. (G) Wild-type MEFs were transfected with siRNA targeting BUBR1 or luciferase (Luc) mRNA for 6 days, after which the transfected cells, along with parental MEFs, were fixed and stained with antibody to α-tubulin and with DAPI. Polyploid cells were scored from 500 MEFs. Error bars represent means ± standard deviation.