Proteasome glo chymotrypsin like cell based assay reagent

H2452 cells were seeded on a 96-well white plate (5 × 10³ cells/100 μL/well), cultured for 24 h, and subsequently treated with bortezomib 50 nM or bortezomib 50 nM +TP 20 µM, T3 20 µM, TOS 20 µM, T3E 20 µM for 6 h, and another 6 h except for bortezomib. After the treatment, to assess chymotrypsin-like activity in cells, 50 µL of Proteasome-Glo™ Chymotrypsin-Like Cell-Based Assay Reagent (Promega Japan, Tokyo, Japan) was added to each well, and the plate was then incubated at room temperature for 20 min. The chymotrypsin-like activity was assessed based on estimated chemiluminescence intensity using a luminometer (Infinite M1000 PRO, TECAN Japan).

H2452 cells were seeded on a 96-well white plate (5 × 10³ cells/100 µL/well), cultured for 24 h, and subsequently treated with α-T3E (20 µM) or vehicle for 24 h. After the treatment, to assess the chymotrypsin-like activity in cells, 50 µL of Proteasome-Glo™ Chymotrypsin-Like Cell-Based Assay Reagent (Promega Japan, Tokyo, Japan) was added to each well, and the plate was then incubated at room temperature for 20 min. The chymotrypsin-like activity was assessed based on the estimated chemiluminescence intensity using a luminometer (Infinite M1000 PRO, TECAN Japan).

MDA-231 were treated with various concentrations of fludarabine (10–200 µM) for 24 h with or without 50 U/ml of IFN-γ. Cells were harvested and plated at 2.5×10⁴ cells/well in white-bottom 96-well plates. Bortezomib was added at 50 nM as negative control for 1 h. Proteasome-Glo chymotrypsin-like cell-based assay reagent was added according to manufacturer instructions (Promega). Luminescence was quantified with the Synergy 4 microplate reader (BioTek).