Fig. 4. Separase is required for efficient dissociation of arm cohesion. (A) Experimental design using asynchronous cells. Immortalized fibroblasts derived from separase conditional-knockout mouse embryos were infected with adenovirus encoding Cre recombinase or -galactosidase (lacZ) at MOI 200. After 2 days, mitotic cells were collected following 1 hour of treatment with 100 ng/ml nocodazole (or with 25 µM MG132, as a control; data not shown) and incubated in the presence of nocodazole (or MG132) for another 0.5, 1 or 3 hours, when cells were fixed and chromosome spreads were stained with Giemsa. (B) Representative chromosome configurations with a normal number of chromatids (two chromatids; left panels) and diplochromosomes (four chromatids; right panels) are shown. Note that chromosomes appear V-shaped when arm cohesion is dissolved, because mouse chromosomes are acrocentric. Insets show magnification images of the chromosomes in the boxed regions. (C) Approximately 200 cells were assessed for arm cohesion and summarized. Note that diplochromosomes (four chromatids) appeared only in Cre-introduced separase-depleted cells, which were scored separately from chromosomes with two chromatids. (D) Experiment designed to enrich the first mitosis after separase depletion. At 10 hours after adenovirus infection, cells were treated with 1 µM aphidicholin for 24 hours to arrest cells at early S phase. At 7 hours after the release from aphidicholin, when many cells were in G2 phase, either 100 ng/ml nocodazole (Noc) or 25 µM MG132 were added and cells were incubated for 2-4 hours. Mitotic cells were collected and analyzed by chromosome spreading. (E) Quantification data of arm cohesion from the experiment in D. Approximately 200 cells with two chromatids were assessed for each time-point.