Bi2536

All cell lines were cultured in a 5% CO₂ humidified atmosphere at 37°C. HEK293T (female, human embryonic kidney epithelial), hTERT RPE-1 (female, human epithelial cells immortalized with hTERT), and A375 cells (female, human malignant melanoma epithelial) are from ATCC. hTERT RPE-1 and A375 stably expressing Cas9 were from D Durocher (Hart et al., 2015 (link); Zimmermann et al., 2018 (link)). All references to RPE-1 and A375 cells herein refer to hTERT RPE-1 or A375 stably expressing Cas9. RPE-1, HEK293T, and A375 were grown in Dulbecco’s modified Eagle’s medium (Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco) and 2 mM Glutamax (Gibco). PLK4 inhibitor centrinone B (Tocris) was used as described. Nutlin-3a (Cayman Chemical) was used at 600 nM. The CDK1, PLK1, and Aurora A kinase inhibitors RO-3306 (Selleck Chemicals), BI-2536 (ChemieTek), and MLN8237 (Selleck Chemicals) were used at 10 μM, 100 nM, and 200 nM, respectively. MG132 (Selleck Chemicals) was used at 10 μM. G418 (WISENT Bioproducts) was used at 600 μg/mL for cell selection and 200 μg/mL for routine culture. All cell lines used have been authenticated by STR profiling and tested negative for mycoplasma contamination.

A number of anti-neoplastic drugs were tested for their ability to inhibit growth of MM cells: the tyrosine kinase inhibitors (TKI) bosutinib, dasatinib, imatinib, sorafenib, sunitinib, and nilotinib, the ErbB-receptor inhibitors lapatinib, erlotinib, and gefitinib, the Aurora-kinase inhibitor VX-680, the HSP90 inhibitor 17AAG, the PLK-1 inhibitor BI2536, the pan-BCL-2 antagonist obatoclax, and the HDAC-inhibitor vorinostat were purchased from Chemietek (Indianapolis, IN, USA). The PI3 kinase/mTOR inhibitor BEZ235 was obtained from Selleck Chemicals (Houston, TX, USA). Stock solutions of drugs were prepared by dissolving in dimethylsulfoxide, DMSO (Merck, Darmstadt, Germany). RPMI 1640 medium and fetal calf serum (FCS) were purchased from PAA Laboratories (Pasching, Austria), and ³H-thymidine from PerkinElmer (Waltham, MA, USA). FITC-labeled CD34 monoclonal antibody (mAb) 581, PE-labeled CD34 mAb 581, FITC-labeled CD138 mAb MI15, PE-labeled CD138 mAb DL-101, PerCP-labeled CD45 mAb 2D1, APC-labeled CD38 mAb HIT2, PE-labeled and Alexa Fluor® 647-labeled active caspase-3 mAb C92-605 were purchased from BD Biosciences (San Jose, CA, USA). The PerCP-labeled CD20 mAb 2H7 and the APC-labeled CD27 mAb O323 were obtained from Biolegend (San Diego, CA, USA), and an Annexin V/FITC kit from eBioscience (San Diego, CA, USA).