Bortezomib

Stock solutions for platinum-based agents (10 mM cisplatin and carboplatin) and a proteasome inhibitor (1 mM bortezomib) were prepared using DMSO (Sigma-Aldrich; stored at −80 °C), further diluted in 1xPBS to the appropriate concentration, and plated in 96-well PCR plates (VWR; stored at −20 °C). The pharmaceutical compounds were screened at nine concentrations (2–1024 µM cisplatin/carboplatin and 1–10,000 nM bortezomib) using a 2-fold dilution series with matched DMSO concentration vehicle controls. The pharmaceutical compounds were at room temperature (18-25 °C) when added to cells. Proteasome activity was assessed using the Proteasome-Glo Chymotrypsin-like assay (Promega) with bortezomib-treated cells seeded in 96-well clear, flat-bottom microplates (Corning Life Sciences) at a density of 7.5 × 10³ cells per well in 100 µl culture medium (RPMI or DMEM basal medium supplemented with 5%, 10% or 15% FBS or without FBS, and HuMEC Basal Serum-Free medium supplemented with epidermal growth factor, hydrocortisone, isoproterenol, transferrin, insulin, and bovine pituitary extract (Life Technologies)).