4.4.2. Cell Culture and CCK-8 Cell Viability Test

The human cervical cancer cell line HeLa (HPV 18 positive) was purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in DMEM with 10% FBS, penicillin (100 units/mL), and streptomycin (100 μg/mL), in an incubator at 37 ◦C with 5% CO2. The CCK-8 test was performed as previously described [17,41]. Briefly, HeLa cells (1 × <sup>10</sup><sup>5</sup> cells/mL) were cultured in DMEM with different doses of genistein and solvent control for 24–48 h. CCK-8 was then added to the culture medium and was incubated for 1 h. The absorbance was measured at a wavelength of 450 nm (OD450 nm) in a microtiter plate reader (BioRad, Hercules, CA, USA).

#### 4.4.3. Colony Formation Assay

Cells (1 × <sup>10</sup><sup>3</sup> cells/well) were initially grown in DMEM with or without different concentrations of genistein and solvent control. After 7–10 days, the cells were fixed with PFA and stained with crystal violet. Images of five randomly selected fields were captured under an inverted microscope (Nikon, Tochigi, Japan).

#### 4.4.4. Adhesion Assay

This assay was performed according to previous protocols [60,61]. Cells (1 × 105 cells/mL) were grown for 2 h and then cultured with or without genistein (0–100 μM) for another 24 h in a 12-well plate. Cells were collected and reseeded with the concentration of <sup>1</sup> × <sup>10</sup><sup>5</sup> cells/mL in a 96-well plate for 3 h. Afterwards, cells were washed, fixed with PFA, and finally stained with crystal violet. Adhesion was assessed at OD570 nm using a microplate reader. Images of five randomly selected fields were captured under an inverted microscope. The percentage of adherent cells was calculated from the OD values of the genistein-treated group (relative to the OD values of the control group).

#### 4.4.5. Wound-Healing Mobility Assay

This assay was performed according to previous protocols [17]. Briefly, cells (1 × <sup>10</sup><sup>5</sup> cells/well) were grown in a 6-well plate for 24 h to achieve 90% confluency. The medium was discarded and cell monolayers were scratched with a sterile P200 micropipette tip. After the debris was washed away, cells were allowed to grow in serum-free medium with different doses of genistein and solvent control for 24 h. Images of the wound areas were captured at 0 h, 8 h, and 24 h. Cell migration was calculated by measuring cell distances from the wound edges in each treatment group. Cell migration (as a percentage) was calculated based on the migration distances in the genistein-treated group (relative to those of the control group).
