PBK is essential for proliferation of MDA-MB-231 cells. A and B, the PBK shRNA or the vector stable cell line was established in MDA-MB-231 cells using the tetracycline-induced lentiviral expression system. Expression of the shRNA was induced by doxycycline (1 μg/ml) for 3 days. The knockdown of PBK was detected by immunoblotting (in A), and effects of PBK knockdown on the cell proliferation was examined by cell counting with a hemacytometer (in B). Data are from at least three independent experiments. C and D, effects of the PBK inhibitor HI-TOPK-032 on MDA-MB-231 cell proliferation were observed and photographed under a phase microscope (in C) and the proliferation rate was quantified by cell counting with a hemacytometer (in D). The variation within the triplicate is indicated with the error bars. The proliferation rate is calculated using the formula PR = (Nt – No)/No, where PR stands for proliferation rate, No for the cell number at seeding, and Nt for the cell number at t time point after seeding.