Hsp72-T66E rescues mitotic defects that arise upon Nek6 depletion. (A) HeLa cells were mock or Nek6 depleted for 48 h before transfection with Flag-Hsp72 constructs as indicated for 24 h and IF with Flag and phospho-H3 antibodies. The mitotic index of transfected cells was counted by microscopic analysis of chromatin. (B) HeLa cells transfected with Flag-Hsp72 constructs indicated for 24 h were stained with Flag and α-tubulin antibodies and scored by IF for the occurrence of nuclear defects. Data in A and B are means (±SD) of 100–300 cells. (C) HeLa cells were treated as in A, placed on ice for 10 min, and analyzed by IF with antibodies against α-tubulin to reveal K-fibers (green) and Flag to detect transfected cells. (D) Total K-fiber intensity from C is plotted relative to that in mock-treated samples. (E and F) HeLa cells were treated and processed as in C but with ch-TOG (E) and TACC3 (F) antibodies. (G and H) Total ch-TOG and TACC3 intensity from E and F is plotted relative to that in mock-treated samples. Error bars show means ± SD. In C, E, and F, DNA was stained with Hoechst 33258 (blue). siMock, mock siRNA; WT, wild type. *, P < 0.05. Bars, 10 µm.