Fig. 2.

HSP70 is required for assembly of a bipolar mitotic spindle. a, b Inhibition of HSP70 induces spindle defects. CGL2 cells were treated with PES at the indicated concentrations for 24 h and were then fixed and stained for mitotic spindles with antibodies against α-tubulin (red) and γ-tubulin (green). The chromosomes were counterstained with DAPI (blue). The percentage of mitotic cells containing abnormal mitotic spindles was determined using at least 500 mitotic cells from three independent experiments. c PES induces mitotic arrest. Cells were treated as in A and then fixed for analysis of cell cycle distribution. The results (mean ± SD) are from four independent experiments. d PES induces cell death. Cells were treated with PES at the indicated concentrations for 72 h, and then subjected to viability assays. The results (mean ± SD) are from three independent experiments. e Expression level of HSP70 in cells stably depleted of HSP70 (sh-HSPA1A/B). Control (sh-Luc) and sh-HSPA1A/B cells were untreated or treated with arsenic trioxide (ATO) or PES for 14 h; the expression of HSP70 was then examined by immunoblotting. f, g Decreased accumulation of HSP70 at the spindle pole in cells stably depleted of HSP70 (sh-HSPA1A/B). Logarithmically growing control (pLKO.1) and sh-HSPA1A/B cells were fixed and immunostained for HSP70 (green) and α-tubulin (red). Relative HSP70 intensity at the spindle pole was determined (median ± 25 percentiles) from three independent experiments. *p < 0.05 by Mann–Whitney rank sum test compared to the control transduced cells (pLKO.1). h, i Depletion of HSP70 induces spindle defects. Control cells (sh-Luc) and cells depleted of HSP70 (sh-HSPA1A/B) were fixed and stained for mitotic spindles as in A. The percentage of mitotic cells containing abnormal mitotic spindles was determined using at least 500 mitotic cells from three independent experiments. Cells stably depleted of HSP70 (sh-HSPA1A/B) were further transduced with a CMV promoter-driven wild type HSP70 (HSP70-wt) or a deletion mutant (HSP70-d5). pFB-Neo is an empty vector and serves as a control. *p < 0.05 by Student’s t test compared to the control depleted cells (pLKO.1). ^# p < 0.05 by Student’s t test compared to the no ectopic HSP70 control