Barasertib

AURKB was inhibited in RB cells by treating them with commercially available AURKB inhibitors: barasertib (AZD1152-HQPA), GSK1070916, and ZM447439 (Apex Bio, Houston, TX, USA). Cell viability was performed using the trypan blue dye (Sigma-Aldrich) exclusion method. The decrease in cell viability in response to inhibitor treatment or shRNA-mediated knockdown was compared with untreated or scrambled controls.

HeLa cells were cultured in DMEM (Corning), 10% fetal bovine serum (Life Technologies), and 1% 100× penicillin/streptomycin solution (Corning) at 37°C, 5% CO₂, and 85% relative humidity. Mitotic HeLa lysates were prepared by dosing cells at ∼70–80% confluency for 16 h with 3 μM STLC and then isolating mitotic cells via vigorous shake-off. The mitotic cells were washed three times with cold PBS (Corning) supplemented with STLC. After the removal of the final wash, protease inhibitors (leupeptin, pepstatin A, and chymostatin, 10 μg each as a 10 mg/ml mixture of all three in dimethyl sulfoxide [DMSO]), cytochalasin D (10 μg from a 10 mg/ml solution in DMSO), and nocodazole (7.5 μg as a 25 mM solution in DMSO) were added to the pellet, which was subsequently frozen in liquid nitrogen and thawed at room temperature three times to lyse cells. The lysate was then spun at 20,000 × g for 30 min at 4°C and the supernatant immediately used in experiments. For cells treated with okadaic acid (1 μM; Enzo) or barasertib (1 μM; ApexBio), the drug was added to the final wash of the cells and also just before lysis after removal of the final PBS wash.