5. MTT cell proliferation assay

MTT cell proliferation assay was performed as described [18]. ESCC cells (2,000 to 8,000 cells/well) were seeded in a 96-well plate overnight before treatment with curcumin (1.25 to 20 μM) and its analogues (0.625 to 5 μM) for 48 hours. MTT reagent (Sigma-Aldrich) was added and incubated for an hour. The reduced MTT was dissolved in sodium dodecyl sulfate. Colorimetric signal was measured at 570 nm, with 650 nm as a reference wavelength, in a Multiskan FC microplate reader (Thermo Fisher Scientific) and analyzed using Skanlt software ver. 3.1 (Thermo Fisher Scientific). Experiments were performed in triplicate wells, and repeated for three times.