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Fig. 1. Aurora B activity is required for loading condensin I in prometaphase. (A) HeLa cells stably expressing EGFP-tagged kleisin-
(condensin II) were enriched in S-phase by a double-thymidine arrest and synchronously released into mitosis. The levels of EGFP-kleisin- associating with chromatin were monitored over time as cells entered mitosis. Chromosomes were visualised by adding the DNA-intercalating dye Hoechst 33342 (0.2 µg/ml). Aurora B activity was compromised either by adding Hesperadin (100 nM) 1 hour before the bulk of the cells entered mitosis or by depleting the protein with siRNAs specific to Aurora B for 30 hours. Movies were taken on an Olympus DeltaVision microscope (63x objective, three slices every 1 minute, 1x1 binning). Projected images are shown. (B) Quantification of chromatin-bound EGFP-kleisin- in control, Hesperadin-treated and Aurora B siRNA-transfected cells over time. Intensities were quantified using the Hoechst signal as a reference. The average cytoplasmic intensity was subtracted as background intensity. Averages of seven cells (n=7) for each condition are shown. Time point 0 (t=0) corresponds to nuclear envelope breakdown (NEBD). (C,D) Same experiment as in A and B using a HeLa cell line stably expressing kleisin-
tagged with EGFP (condensin I). Curves represented in D are averages from seven cells (n=7) for each condition.
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