Cell proliferation assay/Cell Counting Kit (CCK)-8 assay

The proliferation of the HCT116 cells and FHC cells was measured using a CCK-8 assay (Dojindo Molecular Technologies, Inc.). There were 3 general groups: The blank group (McCoy's 5A medium without cells or drugs), the control group (cells were cultured in McCoy's 5A medium without the drug) and the experimental groups (cells were exposed to 2.5, 5, 10, 15, 20, 25, or 50 μM morusin for 72 h, or to 9.1, 18.2 or 36.4 μM morusin for 24, 48 or 72 h). Following treatment, the culture medium was discarded and 100 μl of fresh medium including 10 μl of CCK-8 was added to each well. Following 4 h of incubation in a humidified 5% CO₂ atmosphere at 37°C, the absorbance was measured at 450 nm using a Multiskan Spectrum Microplate Reader (Bio-Rad Laboratories, Inc.). The experiments were performed in triplicate. The percentage inhibitory rate (IR%) of the treated cells was calculated using the following formula: IR%=1-[(EXP group OD450 nm-blank group OD450 nm)/(CTRL group OD450 nm-Blank group OD450 nm)] x100%.