Fig. 5
HSP70 facilitates PLK1 accumulation at the spindle pole and ameliorates PLK1 interference-induced spindle abnormalities. A Representative images of PLK1 or p-T210-PLK1 (green) at the spindle pole specified by γ- or α-tubulin (red) in pLKO.1- or shHSP70-transduced cells. B Relative PLK1 and p-T210-PLK1 intensity at the spindle pole (SP) with numbers of examined spindle poles (n) indicated. Mann–Whitney rank sum test was performed. *p < 0.05 comparing shHSP70 to pLKO.1. C GSD images of PLK1 accumulation at the spindle pole, specified by α-tubulin (α-tub, green), in control or HSP70-depleted cells. Spindle pole regions in the wide-field images are indicated with red boxes (upper panel), and magnifications are shown in the GSD images (lower panel). D PLK1 intensity at the spindle pole (SP) was determined from GSD images and numbers of examined spindle poles (n) are indicated. Mann–Whitney rank sum test was performed. *p < 0.05 comparing shHSP70 to pLKO.1. E Representative images of pFB-Neo—(a) and FLAG-HSP70—(b) transduced cells, untreated or treated with 10 μM BI2536 for 30 min before fixation, and immunostained with PCNT (green) and α-tubulin (red). F Relative PCNT intensity at the spindle pole (SP) with numbers of examined spindle poles (n) indicated. Mann–Whitney rank sum test was performed. #p < 0.05 comparing BI2536 to untreated; *p < 0.05 comparing FLAG-HSP70 to pFB-Neo. G Percentages of cells with abnormal spindles. #p < 0.05 comparing BI2536 to untreated by Student’s t test; p value is indicated comparing FLAG-HSP70 to pFB-Neo by two-way ANOVA. H Representative images of pLKO.1—(a) and shHSP70—(b) transduced cells, untreated or treated with 10 μM BI2536 for 30 min before fixation, and immunostained with PCNT (green) and α-tubulin (red). I Relative PCNT intensity at the spindle pole (SP) with the number of examined spindle poles (n) indicated. Mann–Whitney rank sum test was performed. #p < 0.05 comparing BI2536 to untreated; *p < 0.05 comparing shHSP70 to pLKO.1. J Percentages of cells with abnormal spindles. #p < 0.05 comparing BI2536 to untreated by Student’s t test; p value is indicated comparing shHSP70 to pLKO.1 by two-way ANOVA