The polar chromosome phenotype in CENP-E–depleted cells can be rescued by expression of a phosphomimetic BubR1 mutant. (A) Schematic representation of transfection, chemical inhibitor treatment, and live-cell imaging protocol. (B–D) Representative still frames of live-cell microscopy of H2B-EYFP cells transfected without (B) or with CENP-E siRNA and mCherry (C) or mCherry-BubR1T608E (D) expression vectors. Arrows point to polar chromosome outside the metaphase plate. The transfected cells were identified by mCherry before live-cell imaging. In C, all 11 filmed cells had polar chromosomes by the end of the filming (300 min). In D, 9 out of 10 filmed cells had all chromosomes aligned at the metaphase plate. Bar, 10 µm. (E–H) Expression of the phosphomimetic BubR1T608E mutant rescues polar chromosome phenotype in CENP-E–depleted cells. Indirect immunofluorescence analysis of CENP-E (E), mCherry-BubR1T608E (F), ACA (G), and DNA (H). Arrows point to polar chromosomes outside the metaphase plate. (I) The percentage of mitotic cells with or without polar chromosomes analyzed in cells transfected with CENP-E siRNA and with or without the mCherry-BubR1T608 construct as indicated (± SD; ≥50 cells each from two repeated experiments were counted). Bars, 10 µm.