Bi2536

CD133⁺ U87 stem cells and CD133⁺ U251 stem cells were assigned to Blank group, control group, PLK1 inhibitor BI2536 group (treated with 0.5 nmol/L BI2536, Selleck Chemicals, Houston, TX), PLK1 inhibitor Volasertib group (treated with 0.5 nmol/L Volasertib, Selleck Chemicals), pcDNA3.1 group, pcDNA3.1‐PLK1 group (cells transfected with PLK1‐pcDNA3.1), si‐NC group, PLK1‐siRNA1 group (cells transfected with PLK1‐specific siRNA1) and PLK1‐siRNA2 group (cells transfected with PLK1‐specific siRNA2). The sequence of PLK1 siRNA is listed in Table 1. The oligonucleotides were purchased from Gene PharmaCo., Ltd. (Shanghai, China). U87 and U251 stem cells were plated in antibiotic‐free medium. Then, the medium was changed to serum‐free Opti‐MEM. Transfection was performed under the guidelines of Lipofectamine 2000 (Invitrogen Inc.).

To avoid potential unspecific effects of the drugs conventionally used for cell synchronization on the centriole cycle, cells were synchronized exclusively by mitotic shake off. Mitotic cells were collected by gently tapping on the tissue culture flasks containing logarithmically growing cells and replated on the coverslip or in the fresh dish, according to the needs of a particular experiment. One hour after replating entire culture was synchronously in G1. These cells were then either cultured without any further treatment and analysed at appropriate time points, or treated with 2 mM HU (Sigma) to induce S-phase arrest, or with 9 μM Cdk1 inhibitor RO-3306 (Tocris Biosciences) to induce G2 arrest10 (link). Plk1 inhibitor BI2536 (200 nM)39 (link) (Selleckchem) was added to some samples to inhibit endogenous Plk1 activity, when needed. If left untreated, G1 cells progress synchronously through the cell cycle allowing unambiguous determination of the cell cycle phase. Examination of the cells under bright light by differential interference contrast microscopy was used to select cells in a particular phase of mitosis for further imaging or fixation, as illustrated in Supplementary Fig. 7.