MTT Assay

The cell viability was measured using the MTT assay kit (ATCC, Inc., Manassas, VA), and the manufacturer’s protocol was followed. Briefly, 1000 cells in 100 μl were plated in each well in a 96-well plate and incubated overnight. On the next day, the cell medium was replenished, and various concentrations of AZA were added to each well (triplicate) accordingly and incubated at 37°C for 72 hours. After incubation, 10 μl of MTT reagent was added to each well, and the plate was incubated at 37°C for 4 hours. A total of 1000 μl of detergent reagent was then added to each well, and the plate was left at room temperature in the dark for 4 hours. The optical density of absorbance at 570 nm was recorded using a Synergy2 multimode microplate reader (Biotek, Inc., Winooski, VT). The cell viability was calculated based on the optical density value normalized to blank control. The IC₅₀ of AZA in 231 and 231 Br cells was calculated based on the cell viability measured by three independent MTT assays.