Eg5-G268V robustly bundles spindle MTs and is essential for spindle assembly in KIRC-2 and -3 cells. (A) Eg5-G268V bundles MTs in high salt buffers. Squashes from reactions containing XR-MTs (grayscale) alone or in combination with Eg5-WT or Eg5-G268V. Reactions contained 50, 150, or 300 mM KCl as indicated. Bar, 40 µm. (B) Eg5-G268V-EGFP bundles MTs in interphase cells. Single optical sections of HeLa cells expressing Eg5-WT-EGFP or Eg5-G268V-EGFP (grayscale/green) and stained with antibodies targeting tubulin (pseudocolored purple). LUTs for individual channels are scaled identically. Bar, 40 µm. (C) Spindle MTs are unusually bundled in KIRC-2 and -3 cells. Maximum intensity z-projections of indicated cell types 2 d after transfection with nontargeting (Control) or Nuf2-targeting (-Nuf2) siRNAs and stained with antibodies targeting centromeres (CREST, red) and tubulin (grayscale and green). DNA, blue. Bar, 10 µm. (D) Eg5 depletion induces a mitotic arrest in KIRC-2 and -3. Bright-field images of indicated cell types 1 d after transfection with nontargeting (Control) or Eg5-targeting (-Eg5) siRNAs. Bar, 200 µm. See also Fig. S2.