Protein Expression Analysis in Hepa1c1c7 Cells

The analysis of protein expression were performed as previously published [15 (link), 16 (link)]. In brief, the Hepa1c1c7 cells (1 × 10⁵ cells/well, the cell density reached 80 to 90% confluence), were treated with PHO-S (0.3–2.0 mM), DODAC/PHO-S 1:1 (0.3–2.0 mM), and empty DODAC (0.3–2.0 mM), for 12 h, were washed with PBS and resuspended in FACS buffer with 2.5% paraformaldehyde for 1 h. After washing, cells were again resuspended in a primary antibody specific for the proteins anti-CD44, anti-CD90, anti-p53, anti-p21, anti-p27, anti-Bax, anti-Bcl-2, anti-caspase-3, anti-caspase-8 (Abcam, Cambridge, MA, United States); anti- cytochrome c, anti-DR4 (Santa (Cruz Biotechnology Inc., Santa Cruz, EUA) and anti-cyclin D1 (Cell Signaling Technology, Danvers, MA), at a concentration of 1 μg/ml at 4 °C, for 1 min. The corresponding isotope antibody was used as a negative control and as a secondary antibody was used Goat anti Mouse IgG (H/L): FITC (AbD Serotec, Raleigh, NC, United States). The cells were pelleted, washed twice with PBS, then, fluorescence-activated cell sorting (FACS) analysis was performed on BD Biosciences FACs Calibur flow cytometer (Becton Dickinson, San Jose, CA, United States) using Cell Quest and Win MDI 2.9 softwares.