96 well flat bottomed plates

The chymotrypsin-like activity of the proteasome after treatment with the above-mentioned proteasome inhibitors was measured using the luminescence-based Proteasome-Glo assay (Promega). Cells were plated in 96-well flat-bottomed plates (Perkin Elmer), cultured for 24 h and then incubated for 2 h with the respective compounds at indicated concentrations or DMSO-only solvent control. A peptide substrate for the chymotrypsin-like activity of the proteasome conjugated to recombinant luciferase, Suc-LLVY-(succinyl-leucine-leucine-valine-tyrosine-)aminoluciferin, was added for 10 minutes, before luminescence was measured with a BioTek Synergy 2 Luminometer (BioTek). Data were normalized to the DMSO-only control group.

Apoptosis and cell viability studies were performed using the Caspase-Glo and CellTiter-Glo luminescence-based assays (Promega) [41 (link)]. Cells were plated in 96-well flat-bottomed plates (Perkin Elmer) after transfection and incubated for 48 hours (Caspase-Glo) or 72 hours (CellTiter-Glo). Luminescence was measured with a BioTek Synergy 2 Luminometer (BioTek). Data were normalized to the cells transfected with non-targeting control siRNA.