Methanol

Cells were seeded into 6-well plates at a density of 1,000/well, grown overnight and then treated with osthole (0, 50, 100, or 150 μM) for 12 days. At the end of the experiments, the cells were fixed with methanol (Sangon Biotech Co., Ltd., Shanghai, China) for 20 min and stained with 0.5% crystal violet (Sangon Biotech). Cell colonies with ≥50 cells were counted using an inverted microscope (Leica Microsystems GmbH, Wetzlar, Germany) in five randomly selected fields.

After transfection, (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) MTT assay was carried out to assess cell proliferation. Briefly, 100 μl of the transfected CaOV3 and SKOV3 cells was resuspended and seeded into 96-well plates at a concentration of 1 × 10⁴ cells per well. Then, 50 μl of MTT reagent that was dissolved in PBS was respectively provided into each well at 0, 12, 24, 48, and 72 h after transfection, and the mixtures were maintained in a 37°C incubator for another 4 h. Thereafter, 150 μl of dimethyl sulfoxide (DMSO) was supplied into each well to dissolve the formazan. The optical density (OD) at 570 nm was measured utilizing a microplate reader (Bio-Rad, Hercules, CA, United States). Cell proliferation ratio (%) was calculated according to the formula listed below: Cell proliferation ratio (%) = OD_{experimental group}/OD_NC × 100%.
After transfection, CaOV3 and SKOV3 cells were resuspended and plated into 6-well plates at a concentration of 1,000 cells per well. Then, the plates were maintained in a humidified incubator under 5% CO₂ and 37°C conditions for 2–3 weeks. Colony fixation and staining were respectively conducted utilizing methanol and 0.1% crystal violet (Sangon Biotech). Finally, the images of the colonies were photographed with a microscope (Olympus, Tokyo, Japan).