Hsp72 promotes chromosomes congression and mitotic progression. (A) HeLa:EGFP–lamin A/mCherry-H2B cells were either mock-depleted, depleted with siRNAs against Hsp72 for 72 h, or treated with Hsp70i for 4 h, as indicated, before time-lapse imaging. Stills from representative videos are shown with times (minutes) from mitotic entry indicated. (B and C) Quantification of cells from A indicating the time from NEBD to last chromosome congressed (B) and last chromosome congressed to anaphase onset (C). Data represent cumulative frequencies (±SD). (D) HeLa cells were mock depleted, depleted of Hsp72 for 72 h, or treated with Hsp70i for 4 h, as indicated, and then processed for IF with α-tubulin and CENP-A antibodies. (E) Total spindle MT intensity of cells in D was plotted relative to that of mock-depleted cells. *, P < 0.05. (F) HeLa cells treated as in D before IF with α-tubulin antibodies; enlargements show spindle pole regions. (G) Distance between spindle poles in the z axis of HeLa cells treated as in D. 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. (H) HeLa cells stably expressing mCherry-H2B were arrested in mitosis for 4 h with MG132. Time-lapse imaging was used to follow metaphase plate alignment with times (minutes) from addition of DMSO or Hsp70i as indicated. In D and F, DNA was stained with Hoechst 33258. Data are means (±SD) of 100–300 cells. Bars: (A, D, F [right], and H) 10 µm; (F, left) 2 µm.