Determination of apoptosis by flow cytometric analysis

The HepG2 and HCCLM3 cells transfected with si-GACAT3 or si-NC for 48 h were subjected to analysis of apoptosis using Annexin V/propidium iodide (PI), as described previously (18). Liver cell apoptosis was examined using an Annexin V-FITC apoptosis detection kit (Sigma-Aldrich; Merck KGaA) according to the manufacturer's recommendations. Annexin V-positive cells were considered early apoptotic cells, and PI-positive cells were considered late apoptotic/necrotic cells. All liver cell nuclei were counterstained with Hoechst 33342 dye.