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. 2009 Jun 1;185(5):859–874. doi: 10.1083/jcb.200812167

Figure 2.

Figure 2.

Aurora B regulates the localization of hSpindly in response to taxol. (A) HeLa S3 cells were treated with GL2 (control) or Aurora B siRNAs for 40 h, or with ZM447439 (ZM) for 16 h before being stained with anti-hSpindly (red) and anti–α-tubulin (green) antibodies and DAPI (blue). (B) Cells were treated with GL2 (control) or Aurora B siRNAs for 40 h. DMSO (control) or ZM447439 (ZM) was added to GL2-treated cells for the last 16 h. Mitotic cells (nocodazole shake-off) were collected and equal amounts of cell extracts were separated by SDS-PAGE and probed by Western blotting with the indicated antibodies; α-tubulin is shown as loading control. (C) Cells were treated with MG132 for 2 h. During the second hour, taxol was added. Cells were then stained with anti-hSpindly (red) and anti-BubR1 (green) antibodies and CREST serum (blue). (D) Graph showing the numbers of KTs from 10 cells treated as in C (399 KTs in total) that were positive for BubR1 (left circle) or hSpindly (right circle). The overlapping region represents the number of KTs that were positive for both BubR1 and hSpindly. Percentages of KTs positive for BubR1 or hSpindly are shown. (E) Graph showing the percentages of sister KT pairs with both KTs positive (+/+), only one sister KT positive (+/−), or both negative (−/−) for BubR1 and hSpindly, respectively (>90 sister KT pairs from 10 cells were counted). (F) Cells were treated with MG132 for 2 h. During the second hour, DMSO, nocodazole (Noc), or taxol with or without ZM447439 (ZM) was added. Cells were then stained with anti-hSpindly (red) and anti-MAD2 (green) antibodies and CREST serum (blue). (G) Cells were synchronized by sequential thymidine arrest (overnight) and release (9 h) before being treated as in F. Mitotic cells were collected by shake-off. Equal amounts of cell extracts were separated by SDS-PAGE and probed by Western blotting with the indicated antibodies; α-tubulin is shown as loading control. (H) Bar graph showing the quantification of hSpindly and MAD2 staining intensities at KTs (normalized against CREST) of cells in F (20 KTs were counted per cell and error bars indicate the standard deviation [SD] of measurements from 8 cells). Bars, 10 µm.