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. 2010 Nov 15;21(22):3952–3962. doi: 10.1091/mbc.E10-04-0293

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© 2010 by The American Society for Cell Biology

This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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Figure 7. — NM II is required for mitotic spindle formation in HL-1 cells. (A) Immunoblot analysis of NMHC II isoform expression in HL-1 cells. All three isoforms of NMHC II are detected in HL-1 cells. RBL cells and COS-7 cells are used as positive controls for NMHC II-A or NMHC II-B and II-C, respectively. Actin is a loading control. (B) Immunoblot analysis shows double knockdown of NMHC II-A and II-B 72 h after siRNA II-A and II-B treatment. (C) Immunofluorescence confocal images show typical metaphase mitotic spindles (red, β-tubulin; blue, DAPI staining) from control (a), 72 h after NMHC II-A and II-B double siRNA treatment (b and c), and 4 h after 50 μM blebbistatin treatment (d) of HL-1 cells. Simultaneous siRNA NM II-A and II-B knockdown or blebbistatin-treated HL-1 cells develop abnormal spindles. (D) Quantification of abnormal mitotic spindles in HL-1 cells after 72 h of siRNA NMHC II knockdown or 4 h of blebbistatin treatment. Note that simultaneous siRNA knockdown of NM II-A and II-B markedly increases the percentage of HL-1 cells showing abnormal spindles compared with siRNA NM II-A or II-B knockdown alone. (E) Immunofluorescence confocal images of the mitotic spindle from control HL-1 cells stained for NMHC II-A (a and c, green) II-B (b and d, green), and β-tubulin (a and b, red). NMHC II-A and II-B costain with β-tubulin (top, yellow), indicating localization of both NMHC II-A and II-B in mitotic spindles of dividing HL-1 cells, in addition to their cortical localizations (green).