Fig. 2. Cenp-F is required for metaphase chromosome alignment. HeLa cells were transfected with siRNA duplexes then analysed 48 hours later by immunofluorescence. (A) Images of mitotic cells stained to detect Cenp-F (green) and the DNA (red), showing normal prophase, prometaphase, metaphase and anaphase configurations in control cells, and aberrant configurations in Cenp-F-deficient cells; bar, 5 µm. (B) Mitotic cells stained to detect Aurora B (green) and the DNA (red). Control cells show that Aurora B localizes to centromeres (panel i) and spindle midzones (panel ii). In Cenp-F-deficient cells, Aurora B localizes to centromeres but midzones and midbodies are rarely seen; bar, 5 µm. (C) Quantitation of pixel intensities showing that Cenp-F is not required for centromere localization of Aurora B. Values represent the mean±s.e.m. from ten well resolved kinetochores in three different cells. (D) Quantitation showing that repression of Cenp-F reduces the frequency of anaphases to 36%; the values represent the mean±s.e.m. of three independent experiments in which at least 100 mitotic cells were scored per experiment. (E) Proportion of prophases (yellow), prometaphases (red), metaphases (green) and anaphases (blue) following exposure to the proteasome inhibitor MG132; the values represent the mean±s.e.m. of three independent experiments in which at least 100 mitotic cells were analysed per experiment.