Cell viability assay

The cell viability analysis was performed by MTT assay as described previously.25 Briefly, Ec109, KYSE70, and Het-1A cells were seeded in a 96-well plate at a density of 8,000 cells each well and cultured overnight for attachment. The next day, the cells were treated with varying concentrations of CDDO-Me (0–5 μM) for 24 and 48 hours. Following drug treatment, the cells were incubated with 5 mg/mL MTT (10 μL/well) for 4 hours at 37°C. The reaction was terminated by the addition of 100 μL DMSO. The absorbance was measured using a Synergy™ H4 Hybrid microplate reader (BioTek Inc., Winooski, VT, USA) at a wavelength of 450 nm. The absorbance values were normalized by assigning the value of the control line in the medium without drug to 1.0 and the value of the no-cell control to 0. The half inhibitory concentration (IC₅₀) was defined as the CDDO-Me concentration required for decreasing the cells to 50% of the control value. Experiments were performed in at least triplicate.