Enzyme labeled instrument

The MTT assay was done to evaluate the function of At-EE on the proliferation of ovarian tumor cells. In 96-well plates, 1 × 10⁴ cells were seeded into a well with 200 μl medium. Then, various concentrations of At-EE were added into each well. 48 h later, the MTT solution was added into each well and incubated for 4 h at 37°C in a humidified incubator with 5% CO₂ and 95% air. Each well was measured by enzyme-labeled instrument (Thermo) at 570 nm absorbance.

A Cell Counting Kit-8 (CCK-8) assay (Dojindo, Kumamoto, Japan) was used according to the manufacturer’s instructions to measure the effects of drugs on the proliferation of papillary thyroid carcinoma cells. Briefly, BCPAP cells were seeded in 96-well cell plates (Corning Inc., Corning, USA) at a density of 1000 cells/well in 200 μL of culture medium and grown overnight. The next day, vemurafenib or/and E3330 were added to each well at an appropriate concentration, and cells were incubated for 0, 24, 48, 72, and 96 h. Then, 20 μL of CCK-8 reagent was added to each well and incubated for 3 h at 37 °C. Finally, the absorbance was measured with an enzyme-labeled instrument (Thermo) at 450/650 nm. The experiments were repeated at least three times.
For colony formation analysis, BCPAP and K-1 cells were seeded in 6-well cell plates (Corning Inc., Corning, USA) at a density of 500 and 1000 cells/well in 2 mL of culture medium, respectively, and then treated with vemurafenib or/and E3330 at an appropriate concentration for 24 h. In the drug treatment groups, the medium was replaced with a fresh medium after 14 days. Colonies were washed with phosphate-buffered saline (PBS) 3 times, fixed with 4% paraformaldehyde for 20 min, stained with 0.5% crystal violet for 15 min at room temperature, and then counted.

After transfection 12 h with lentiviral particles, CD4⁺ T cells were plated in a 96‐well plate and incubated with anti‐CD3 and anti‐CD28 monoclonal antibodies for 5 days. The cells were treated with 10 μL of CCK‐8 solution at 37°C for 4 h. The absorbance was measured at 450 nm with an enzyme‐labeled instrument (Thermo Fisher Scientific). For apoptosis analysis, CD4⁺ T cells were stained with Annexin V‐FITC/PI. Percentages of apoptotic cells were analyzed with a Calibur flow cytometer (Beyotime).