*4.4. MTS Assay*

To determine cytotoxic activity of rHct-S3, cells (1.0 <sup>×</sup> 104) were seeded in 96-well plates ("Jet Biofil", Guangzhou, China) and cultured in 200 μL of complete culture medium for 24 h at 37 ◦C in a 5% CO2 incubator. The cell monolayer was washed with PBS and treated either with PBS (control) or various concentrations of rHct-S3 (0.01 μM–10 μM) in fresh appropriate culture medium for 24 h. Subsequently, the cells were incubated with 15 μL MTS reagent ("Promega", Madison, WI, USA) for 3 h, and the absorbance of each well was measured at 490/630 nm using Power Wave XS microplate reader ("BioTek", Wynusky, VT, USA).

To determine the antiproliferative activity of rHct-S3, cells (0.7 <sup>×</sup> <sup>10</sup>4) were seeded in 96-well plates and cultured in 200 μL of complete culture medium for 24 h at 37 ◦C in a 5% CO2 incubator. The cell monolayer was washed with PBS and treated either with PBS (control) or various concentrations of rHct-S3 (1, 2, and 4 μM) in fresh appropriate culture medium for 24, 48, 72, 96 h. Subsequently, the cells were incubated with 15 μL MTS reagent ("Promega", Madison, WI, USA) for 3 h, and the absorbance of each well was measured at 490/630 nm using Power Wave XS microplate reader ("BioTek", Wynusky, VT, USA).
