HMMR, CHICA, and DYNLL1 are required for normal mitotic progression. (A) HeLa cells stably expressing GFP-tubulin and mCherry-histone H2B were transfected with the siRNA duplexes shown in the figure for 72 h, and the full cell volume was imaged on an Ultraview spinning-disk confocal microscope every minute as the cells passed through mitosis. Maximum intensity projections of tubulin and histone H2B are shown at the times indicated in the figure. Bars, 10 µm. (B) A maximum intensity z-projection of tubulin and histone H2B are shown in the left column. The full 3D dataset was rotated to view the spindle from a plane perpendicular to the chromosomes and this is shown in the right column. (C) The pole-to-pole distance and (D) angle of spindle rotation at the metaphase-to-anaphase transition were calculated as described in Fig. 3 A and are plotted in the graphs. The Mann-Whitney test was used to calculate P-values for comparison of control and experimental samples. Median spindle angle was significantly different (P < 0.0001) in the HMMR- and CHICA-depleted cells, P < 0.001 in the DYNLL1-depleted cells, but not the HURP-depleted cells (P = 0.8006) when compared with the control. Numbers in brackets indicate sample size.