Hsp72 promotes recruitment of ch-TOG–TACC3 complexes to K-fibers. (A) HeLa cells treated as indicated were placed on ice for 10 min before IF with α-tubulin and CENP-A antibodies to reveal K-fibers. (B) K-fiber intensity is plotted relative to that in mock-treated cells. (C) HeLa cells treated as indicated were incubated with 50 µM monastrol for 4 h, placed on ice for 10 min, and then processed for IF with α-tubulin and CENP-A antibodies. (D) The length of K-fibers in the monopolar spindles is plotted. For box and whisker plots, boxes represent the 25th and 75th percentile, the green and white lines represent the medians and means, respectively, and whiskers show the 10th and 90th percentiles. (E and F) HeLa cells treated as indicated were processed for IF with antibodies against α-tubulin and ch-TOG (E) or TACC3 (F). (G and H) The intensity of ch-TOG (G) and TACC3 (H) relative to tubulin from cells in E and F is indicated. (I) K-fiber intensities were measured as in B after Hsp72 or mock depletion and expression of siRNA-resistant Hsp72 constructs. siMock, mock siRNA; WT, wild type. (J) Hsp72 IPs were prepared from mitotic cells treated with or without Hsp70i and analyzed by Western blotting with the antibodies indicated. (K) The amount of proteins precipitated in J is quantified relative to that in DMSO-treated Hsp70 IPs. (L) Flag IPs prepared from mitotic cells induced to express Flag-TACC3 with doxycycline and treated with Hsp70i were analyzed by Western blotting for Flag, Hsp72, and ch-TOG. (M) The amount of proteins precipitated in L is quantified relative to DMSO-treated Flag IPs. In A, C, E, and F, DNA was stained with Hoechst 33258. Data are means (±SD) of 100–300 cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Bars, 10 µm.