Fig. 4. Kinetochore-spindle-attachment defects in the absence of cohesin are not rescued by topologically linking sister chromatids. (A) Fluorescence images showing microtubules (red) and the kinetochore component Ndc80 (green) in spindles formed in mock- or cohesin-depleted extracts that had been treated with 0.3 µM nocodazole to preferentially depolymerize non-kinetochore microtubules so that kinetochore-microtubule attachments could be visualized. Reactions were spun onto coverslips and processed for immunofluorescence 20 minutes post drug addition. Bar, 5 µm. (B) Fluorescence images showing microtubules (red), Ndc80 (green) and DNA (blue) in control (Mock) and cohesin (Cohesin) depleted spindle assembly reactions that had been incubated with a solvent control (DMSO) or the topoismerase II inhibitor etoposide (50 µM) to prevent DNA decatenation. Drug was added after DNA replication, upon induction of mitosis. Bar, 10 µm.