DAPI staining

Morphological changes in the apoptotic cells were assessed by fluorescent microscopy following DAPI staining. Briefly, the HepG2 cells were seeded at a density of 1×10⁵ cells/ml in 6-well plates and grown for 24 h in a 37°C incubator. Following washing once with PBS, the cells were either left untreated (control group) or treated with D-GalN (50 mM), 5% hPH, or 5% hPH + D-GalN (50 mM). Following incubation for 24 h, the cells were fixed in 4% formaldehyde for 1 h, and permeabilized with 0.1% Triton X-100 (Biosesang, Inc., Seongnam, Korea) for 5 min. Subsequently, DNA-specific fluorochrome DAPI (Invitrogen; Thermo Fisher Scientific, Inc.) was applied to each well, following which samples were incubated for 10 min in the dark at room temperature. Finally, the cells were washed three times with PBS and examined using a confocal microscope (Carl Zeiss AG, Oberkochen, Germany).