Annexin 5 pi staining

For cell cycle analysis, non-transfected and NOLC1-transfected HepG2 cells were cultured to 80% confluency, washed in PBS, trypsinized, fixed in 75% ice-cold ethanol overnight, and treated with 20 μg/ml DNase-free RNase A at 37 °C for 30 min. The cells were stained with propidium iodide (PI; 50 μg/ml; Sigma), and flow cytometry was conducted. Cell cycle profiles were analyzed using Multi Cycle AV software. In the apoptosis assay, cells (1×10⁵) from each sample were subjected to annexin V/PI staining (Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions.

Annexin V/PI staining assay was performed according to the supplier’s instructions (Dojindo Molecular Technology, distributed by Microtech, Italy). Briefly, cells treated with the extract at indicated concentrations for 24 h were centrifuged and suspended in Annexin V binding solution at the final concentration of 1 × 10⁶ cells/mL. A total of 100 μL of this suspension was transferred into a new tube, to which 5 μL Annexin V (FITC conjugated) and 5μL propidium iodide (PI) were added. The reaction was carried out for 15 min at room temperature. The results were acquired using FACS Calibur (BD Biosciences). Graphs show the experimental results of biological triplicates.