Bi 2536

The D17 cell line, known to overexpress PLK‐1 and c‐Myc proteins, was selected for inhibition experiments using BI 2536 (Boehringer Ingelheim, Ingelheim, Germany).³² A 10 mM stock solution of BI 2536 was prepared by resuspending the compound in dimethyl sulfoxide (DMSO). D17 cells treated with DMSO were used as control. First, 3 × 10⁵ cells/well were seeded in six wells cell culture plates and were then treated with BI 2536 at 2.5, 5, 7.5, and 15 nM for 12 and 24 h. Morphological changes were evaluated with a Leica AF6000 LX (Leica Microsystems, Wetzlar, Germany) microscope equipped with a Leica DFC350FX digital camera controlled by the LAS AF software (Leica Microsystems). Based on morphological effect, viability assay was performed using CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI) on 1 × 10⁶ cells/well at 2.5, 5, 7.5, and 15 nM for 12 and 24 h. Similarly, caspase activity for apoptosis detection was evaluated using Caspase‐Glo 3/7 Assay System (Promega, Madison, WI) according to the datasheet after 12 and 24 h of treatment at 2.5, 5, and 7.5 nM. The untreated D17 cell line was used as control while the medium was used as blank to subtract background signal. All experiments were performed in triplicate and repeated three times.

CLBL-1 cells were kindly provided by Dr Barbara Rütgen (University of Wien, Austria). The KLR-1201 cell line was developed in the Drs. Angela McCleary-Wheeler and Kristy Richards’ laboratories (Cornell University, Ithaca, NY, USA). Both cell lines were cultured in IMDM supplemented with fetal bovine serum (10% for CLBL-1; 20% for KLR-1201), 1% glutamine, 100 μg/mL penicillin, and 100 μg/mL streptomycin. BI2536 and MZ1 compounds were purchased from Boehringer Ingelheim (Ingelheim am Rhein, Germany) and were dissolved in dimethyl sulfoxide (DMSO) at 10 µM stock solution.