Supplemental Materials

This article contains the following supporting material:

• Supplementary Movie 1 - Chromosome movement during mitosis with a monopolar spindle induced by inhibition of Eg5 using 100 μM monastrol.
• Supplementary Movie 2 - Chromosome movement during mitosis in a Kif2a-deficient cell with a monopolar spindle.
• Supplementary Movie 3 - Chromosome movement during mitosis in a Kif2b-deficient cell with a monopolar spindle.
• Supplementary Movie 4 - Chromosome movement during mitosis in a Kif2b-deficient cell with a disorganized spindle.
• Supplementary Movie 5 - Cytokinesis failure in a Kif2b-deficient cell with a monopolar spindle.
• Supplementary Movie 6 - Cytokinesis failure in Kif2b-deficient cells with a bipolar spindle.
• Supplemental Figure 1 - Kif2b expression in human tissues. Total cell protein from human brain (B), heart (H), kidney (K), liver (Li), lung (Lu), ovary (O), placenta (P), skeletal muscle (Sk), spleen (S), and testes (T) were separated by size by SDS-PAGE and blotted with Kif2b polyclonal serum.
• Supplemental Figure 2 - Expression and localization of Kif2a, Kif2b, and MCAK are independent of each other. (A) Total cell extracts prepared from U2OS cells that were either untreated or transfected with Kif2b-specific siRNA were separated by size by SDS-PAGE and blotted with antibodies specific to Kif2a, MCAK, and actin as indicated. (B) U2OS cells transfected with Kif2b-specific siRNA were fixed and stained for microtubules in green, DNA in blue, and either Kif2a, MCAK or CENP-E in red as indicated. The scale bar represents 5 μm. (C) U2OS cells expressing GFP-Kif2b were untreated or transfected with siRNA specific to either Kif2a or MCAK as indicated. Cells were fixed and stained for microtubules in red, DNA in blue and GFP-Kif2b in green. The scale bar represents 5 μm.
• Supplemental Figure 3 - Kif2a, Kif2b, and MCAK are required for mitotic spindle formation in human hTERT-immortalized RPE-1 cells. (A) Untreated RPE-1 cells (control) or RPE-1 cells transfected with Kif2a-, Kif2b-, MCAK-, or both Kif2a- and Kif2b-specific siRNA were fixed and stained for microtubules in red and DNA in blue. The scale bar represents 5 μm. Percentages of mitotic cells with monopolar spindles are indicated in each panel and are consistent with percentages of monopolar spindles observed in U2OS cells following similar treatments. (B) Total cell extract from untransfected RPE-1 cells (untreated) or RPE-1 cells transfected with siRNA specific to either Kif2a or MCAK were separated by size by SDS-PAGE and blotted with antibodies specific to Kif2a, MCAK, and actin as indicated.
• Supplemental Figure 4 - Chromosome position on monopolar spindles. U2OS cells were either treated with 100 μM monastrol to inhibit Eg5 activity or transfected with Kif2a- or Kif2b-specific siRNA. The area occupied by chromosomes on monopolar spindles in mitotic cells was quantified in three trials of at least 50 cells each. Error bars represent standard deviations.
• Supplemental Figure 5 - Kinetochore localization of GFP-Kif2b relies on CLASP1. (A) Untreated U2OS or U2OS cells transfected with CLASP1-specific siRNA were fixed and stained for microtubules in green, CLASP in red and DNA in blue. (B) Untreated U2OS or U2OS cells transfected with CLASP1- or Nuf2-specific siRNA as indicated were fixed and stained for Hec1 in red, GFp-Kif2b in green, DNA in blue. The scale bar represents 5 μm.