Fig. 5. Scc1 cleavage is required for the complete removal of cohesin from chromosome arms. (A) Induction of Scc1-Myc. Both wild-type and non-cleavable Scc1-Myc could be detected 24 hours after doxycycline addition. Scc1 antibodies recognize both endogenous and exogenous Scc1, as indicated. (B) Experimental protocol. HeLa cells that can inducibly express either wild-type or non-cleavable Scc1-Myc were induced of their expression by the addition of doxycycline for 40 hours. Mitotic cells enriched by 1 hour of treatment with 100 ng/ml nocodazole were collected and further incubated for 3 hours in the presence of nocodazole. (C) Representative images of wild-type and non-cleavable Scc1-Myc-expressing cells (top two panels). After a hypotonic treatment, cells were cytospun onto glass slides and immunostained with antibodies to the Myc epitope (not shown, but red in E) and the condensin I subunit CAP-G (green), and DNA was counterstained with DAPI (blue). Chromosomes in the boxed regions are magnified and shown as an example for arm-open or -closed chromosomes (bottom panels). (D) Quantification results from 100 prometaphase cells for each experiment are summarized in the histogram. The cell population for the open and closed arms is shown in dark grey and white, respectively, and an unclassified population is in light grey. (E) The Scc1-Myc-positive prometaphase and metaphase cells were further classified accordingly to the distribution pattern of Scc1-Myc on chromosomes, as colour-coded. (F) One-hundred prometaphase/metaphase cells were classified into three categories based on Myc-staining pattern, as exemplified in E. An unclassified population is shown in grey. Bar, 10 µm.