hSpindly localizes at outer KTs and spindle poles, downstream of the RZZ and Ndc80 complexes. (A) HeLa S3 cells in interphase were stained with anti-hSpindly (red) and anti–α-tubulin antibodies (green), and DAPI (blue). (B) Cells at different mitotic stages were stained with anti-hSpindly antibody (red), CREST serum or α-tubulin antibody (green), and DAPI (blue). (C) Prometaphase cell stained with anti-hSpindly (red) and anti-BubR1 (green) antibodies and CREST serum (blue). The inset shows a magnification of the selected area (bar, 1 µm). (D) Cells treated for 72 h with GL2 (control) or two independent siRNAs targeting ZW10 (ZW10-1 or ZW10-2 siRNA) were stained with anti-hSpindly antibody (red), CREST serum (green), and DAPI (blue). (E) Western blotting of mitotic (nocodazole shake-off) cells treated for 72 h with GL2 (control), ZW10-1, or ZW10-2 siRNAs. Membranes were probed for the indicated antibodies and α-tubulin is shown as loading control. (F) Cells treated with GL2 (control) or Hec1 siRNAs for 48 h were stained with anti-Hec1 (green) and anti-hSpindly (red) antibodies, and DAPI (blue). (G) Western blotting of mitotic (nocodazole shake-off) cells treated with GL2 (control), or Hec1 siRNAs for 40 h. Membranes were probed for the indicated antibodies and α-tubulin is shown as loading control. Bars, 10 µm.