Fig. 2

Loss of HSP70 function induces spindle pole fragmentation and disrupts the accumulation of PCNT and CEP215 at the mitotic centrosome. A Western blot analysis of the efficiency and specificity of shRNA-mediated depletion of HSP70. The numbers underneath each blot indicated the ratio of expression level of each protein to that in the pLKO.1 control as normalized by the loading control GAPDH. Mean ± SD from three independent experiments. B Types of abnormal spindle formation in HeLa-S3-tub-EYFP cells were visualized by time-lapse imaging, and still images from the indicated times are shown. (a) Cells with more than two visible MTOCs before mitosis entry were regarded as ‘multiple MTOC’. (b) Mitotic cells in which a bipolar spindle was first constructed, but then either pole was split into multiple poles in the later frames were defined as ‘fragmented spindle pole’. (c) Mitotic cells with bipolar spindles that did not split into extra poles but kept rotating the spindle axis with varying orientations throughout the imaging period were classified as ‘misoriented spindle’. C Percentages of control vector-transduced (pLKO.1) or HSP70-depleted (shHSP70) mitotic Hela-S3-tub-EYFP cells with the indicated types of abnormal spindles. *p < 0.05 by Mann–Whitney rank sum test comparing shHSP70 to pLKO.1. The percentage of mitotic cells containing abnormal mitotic spindles was determined using at least 100 mitotic cells from six independent experiments. D Representative images of control or HSP70-depleted interphase and mitotic CGL2 cells immunostained with EB1 (red) and γ-tubulin (γ-tub, green). E Relative EB1 intensity at the centrosome or spindle pole (SP) with number of spindle poles (n) indicated. Mann–Whitney rank sum test was performed. *p < 0.05 comparing shHSP70 to pLKO.1; N.S. no significance. F Representative images of control or HSP70-depleted and untreated or PES-treated CGL2 cells immunostained with PCNT or CEP215 (green). The spindle was revealed by α-tubulin immunostaining (α-tub, red). Cells were treated with 10 μM and 40 μM PES for 4 h and 0.5 h, respectively, before fixation. G Quantification of PCNT and CEP215 volumes at the spindle pole was performed, and values are shown in the box plots with the number of examined spindle poles (n) indicated. *p < 0.05 comparing shHSP70 to pLKO.1 or PES to untreated by Mann–Whitney rank sum test