Facs cytometry

1 * 10⁵ transfected cells were seeded on 6 cm-diameter plates with RPMI-1640 medium containing 10% FBS for 48 h. Then the cells were harvested, washed twice with ice-cold phosphate-buffered saline (PBS), and re-suspended with binding buffer at a concentration of 1 * 10⁶ cells/ml. The cells were then labeled by using an annexin V-FITC/PI staining kit (eBioscience, United States) according to manufacturer’s instructions. The DNA content of labeled cells was analyzed with fluorescence activated cell sorting (FACS) cytometry (Millipore, United States). Experiments were performed in triplicate.

For the CCK-8 assay, HepG2 cells were seeded in 96-well plates at a density of 2,000 cells per well and incubated for 1, 2, 3, 4, or 5 days. Then, 10 μL of CCK-8 (Dojindo Molecular Technologies, Japan) was added to each well, incubated for 4 h, and mixed gently on an orbital shaker for 2 min before the absorbance value (OD) of each well was measured at 450 nm. For the cell cycle assay, the cells were seeded on 6 cm diameter plates with DMEM containing 10% FBS and labeled using a cell cycle detection kit (Sigma, USA) following the manufacturer’s protocols. The DNA content of labeled cells was analyzed with FACS cytometry (Millipore, USA). The experiments were performed in triplicate.

Cells were seeded on 6 cm-diameter plates with RPMI-1640 containing 10% FBS. After treatment, cells were labeled by using a cell-cycle detection Kit (Sigma, United States) and annexin V-FITC/PI staining kit (eBioscience, United States), according to manufacturer’s instructions. The DNA content of labeled cells was analyzed with FACS cytometry (Millipore, United States). Experiments were performed in triplicate.