2.5. Cell Proliferation and Apoptosis Assay

The effect of Rg3 and noncoding RNAs on cell growth was analyzed by a dye-based cell-proliferation assay as previously described [22]. Briefly, 2 × 10³ cells were seeded per well on a 96-well plate and cultured for 24 h. Afterward, the cells were treated with either Rg3 or noncoding RNA and cultured for up to six additional days. After an appropriate culture period, the cells were stained with WST-8 using the Cell Counting Kit-8 (CCK-8) (Enzo Biochem, New York, NY, USA) to measure cell density at OD₄₅₀ using a spectrophotometer. For the apoptosis analysis, 1 × 10⁶ cells were seeded in a 60 mm plate, treated with Rg3 or transiently transfected with siRNA, and cultured for 24 h. After harvesting, 1 × 10⁵ cells were suspended in a 1x binding buffer provided with the Annexin V-FITC Apoptosis Detection kit II (BD Bioscience, San Jose, CA, USA), then stained with FITC Annexin V(BD Bioscience) and PI (Sigma-Aldrich, St. Louis, MO, USA). Fluorescence was detected with a BD Accuri C6 flow cytometer (BD Bioscience), and the data were analyzed with the BD Accuri C6 software (BD Bioscience). Cell-cycle analysis was performed using a flow cytometer as previously described [23]. The cell-proliferation index was calculated using the following formula: proliferation index = (S+G2+M)/(G0/G1+S+G2+M) × 100 (%), where each letter represents the number of cells at each stage.