Cell Viability Assay

Cell viability was determined by the MTT assay following the vendor’s protocol (Amresco, Cleveland, OH, USA). The SMMC-7721 cells were seeded at the initial cell density of 5 × 10³ cells/well in 96-well plates and cultured with varying concentrations of DFP or VC for specified times. At harvest time, the MTT reagent (0.5 mg/mL) was added to all wells in the 96-well plate, followed by incubation for 4 hours. Following the incubation, the supernatant was removed from the wells and dimethyl sulfoxide was added to each well to dissolve the formazan crystals. The absorbance was then measured using a spectrophotometer microplate reader (Bio-RAD, Hercules, CA, USA) at a wavelength of 492 nm. The cell inhibitory rate was expressed as a percentage (%) of 1 minus the ratio of viable cell number after drug treatment relative to the control cell number. The IC₅₀ (50% inhibition concentration, defined as the drug concentration resulting in 50% inhibition vs the untreated culture) of DFP and VC was calculated using the data generated from the MTT experiment. This experiment was repeated 3 times, and the results were statistically analyzed using the unpaired Student’s t test.