Apoptosis Assay

Apoptosis analysis of both SMMC-7721 and HepG2 cells was conducted using the Annexin V-FITC Apoptosis Detection Kit according to the manufacturer’s instructions (Invitrogen, CA, USA). Briefly, cells (2 × 10⁶ cells/dish) were seeded into six-well plates. Following 24-h treatment with and without SSd (5.0 mg/ml), cells were removed from the plates using trypsin, washed with ice-cold PBS twice, and harvested. The cells were then resuspended to approximately 1 × 10⁶ cells/ml and stained with Annexin V-APC and propidium iodine according to the manufacturer’s instructions (KeyGEN BioTECH). Annexin V-APC/PI binding was analyzed by flow cytometry using a BD FACSCalibur system. Each histogram was constructed with the data from at least 5,000 events. All the samples were analyzed in triplicate.