Proteasome glo chymotrypsin like assay

The Proteasome-Glo chymotrypsin-like assay (Promega; catalogue no. G8622) luciferin detection reagent (comprising luciferase enzyme in Proteasome-Glo buffer) was prepared according to the manufacturer’s protocol (29 ). A 1,200 μM (600 μM final assay concentration) and a 40 μM (20 μM final assay concentration) solution of Proteasome-Glo chymotrypsin-like reagent were prepared using the luciferin detection reagent as a diluent, and the mixtures were incubated at room temperature for 60 min. Partially purified stock T. cruzi proteasome solution was diluted 1 in 4 (CMF = 0.25) using proteasome buffer, and 4 μl of the diluted solution was added to various concentrations of oprozomib (Selleckchem; catalogue no. S7049) (12 final assay concentrations ranging from 3.09 × 10^–5 M to 1.11 × 10^–9 M at 1-in-3 dilution increments). Next, 4 μl of either 40 μM or 1,200 μM Proteasome-Glo chymotrypsin-like reagent was added to initiate the biochemical reaction. The reaction was allowed to proceed at room temperature for 60 min, after which the luminescence was read. Data were acquired from 3 independent replicates (n = 3) and processed as described above for the cell-free pIC₅₀ determination experiments.

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)).