HSP70 is required for the proper assembly of pericentriolar material and function of mitotic centrosomes

HSP70 accumulates at the spindle pole in a PLK1-dependent manner and colocalizes with PCNT, CEP215 and γ-tubulin. A Representative images of immunostained HSP70 (green) at the spindle pole in the CGL2 cells. Cells were treated with 20 μM PLK inhibitor III or BI2536 30 min before fixation. Spindle poles were identified by either α-tubulin (α-tub, red) or CEP152 (red) staining. B Relative HSP70 intensity at the spindle pole (SP) was measured. Box plots show the 10th, 25th, 50th, 75th, 90th percentiles, and numbers in parenthesis (n) indicate the number of spindle poles examined. *p？<？0.05, significant decrease compared to untreated by Mann–Whitney rank sum test. C–F PLK1 (C green) and HSP70 (E green) accumulation at the spindle pole (specified by α-tubulin, red) in cells transduced with control vector (pFB-Neo), FLAG-PLK1-WT or D176N. D, F Relative intensity of PLK1 (D) and HSP70 (F) at the spindle pole (SP). Numbers of examined spindle poles (n) are indicated. Mann–Whitney rank sum test was performed. *p？<？0.05 comparing WT to pFB-Neo; #p？<？0.05 comparing D176N to WT. G Colocalization of HSP70 (green) with CEP215 (a), PCNT (b) and γ-tubulin (c) (red) was revealed by 3D-SIM imaging. The mitotic centrosomes in the red-boxed regions in the upper panels were magnified and are presented as wide-field (middle) and SIM (bottom) images. H Percentages of each indicated PCM component that were colocalized with HSP70 with or without HSP70 depletion are shown in the box plot with the number of spindle poles (n) indicated. *p？<？0.05 by Mann–Whitney rank sum test comparing shHSP70 to CGL