Serpinb3

Cells were lysed with RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific), supplemented with cOmplete^™ Protease Inhibitor Cocktail (MilliporeSigma). Proteins were electrophoresed under reducing conditions on 4–15% polyacrylamide gels (Bio-Rad Laboratories, Inc., Hercules, CA) and then transferred onto a nitrocellulose membrane (Bio-Rad Laboratories, Inc.). This membrane was incubated for 60 min with SuperBlock^™ Blocking Buffer (Thermo Fisher Scientific) at room temperature. Incubation with primary antibody was carried out overnight at 4°C. The following primary antibodies were used: SERPINB3 (R & D Systems, Minneapolis, MN, MAB6528; 1:500), SERPINB3 (Thermo Fisher Scientific, PA5-30164; 1:2000), c-Myc (MilliporeSigma, M4439, 1:2000), BBOX1 (MilliporeSigma, WH0008424M1, 1:200), Cas9 (Cell Signaling Technology, Inc., Beverly, MA, 14697T, 1:1000), and β-Actin (MilliporeSigma, A5441, 1:2000). The membrane was incubated with secondary ECL anti-rabbit or anti-mouse IgG HRP-linked antibody (GE Healthcare, Pittsburgh, PA) for 1 hour at room temperature. Protein was visualized using a SuperSignal^™ West Dura Extended Duration Substrate (Thermo Fisher Scientific).

Indirect immunofluorescence was performed on HepG2 and HepG2/SB3 cells seeded on 6-well culture plates as described. Briefly, cells were fixed with methanol:acetone 1:1 (v/v) for 20 min at −20 °C, permeabilized with PBS containing 0.5% Triton X-100 and 0.05% NaN₃ for 10 min (room temperature) and incubated with primary antibodies SerpinB3 (1:100, ThermoFisher Scientific) and c-Myc (1:100, sc-788 Santa Cruz Biotechnology, CA) overnight at 4 °C. Immunopositivity was revealed by means of appropriate Cy3-conjugated antibodies (1:1000 dilution). Nuclei (blue fluorescence) were stained by treating cells with 4,6-diamidino-2-phenylindole (DAPI, 1 mg/ml in methanol) for 30 min at room temperature. Slides were then mounted with ELVANOL (Sigma-Aldrich, St. Louis, MO) and observed under fluorescence microscope (Leica).