Fig. 3. Localization of Sgo1 on chromosome arms. (A) HeLa cells expressing EGFP-tagged Sgo1 were synchronized with thymidine, and, at 9.5 hours after the release, cells were fixed and DNA was counterstained with DAPI. The left panels are representative pictures of prophase, prometaphase and metaphase cells from unperturbed mitosis. The right panels represent cells treated either with 3 µM of an Aurora B inhibitor, ZM447439, 100 ng/ml nocodazole or 25 µM MG132 for 1 hour before fixation. Note that some EGFP signals are discernible on chromosome arms (arrowheads) that are not clearly detectable in nocodazole-treated cells. (B) Total extract of nocodazole-arrested HeLa cells (lane 1) was fractionated into a chromosome-enriched fraction (lane 2) and a cytoplasmic fraction (lane 3). Each fraction was analyzed by immunoblotting with antibodies to Sgo1. Note that these antibodies specifically react with Sgo1 in the chromosome-enriched fraction (left). (Right panels) HeLa cells were transfected with siRNA to Sgo1 during thymidine treatment for 21 hours. At 9 hours after the release, cells were fixed with 4% paraformaldehyde and stained with antibodies to Sgo1. DNA was counterstained with DAPI. Note that signals on chromosome arms (arrowheads) were abolished by Sgo1 depletion (right).