Blocking serum

Cells were fixed with 4% formaldehyde (BioSharp, Hefei, China) for 10 min at room temperature. Cells were then permeabilized with 0.5% Triton X-100 (BioSharp, Hefei, China) for 2 min and treated with a blocking serum (Solarbio, Beijing, China) for more than 5 h. Primary antibody, anti-COUP TF1 (sc-74560, 1:50, Santa Cruz, TX, USA), and anti-Ki67 (ab15580, 1:200, Abcam, Cambridge, UK) were incubated overnight for immunofluorescence. After being washed to remove unbound primary antibodies, cells were incubated with the secondary antibody Dnk pAb to Rb IgG (Alexa Fluor® 488) (ab150074, 1:1000, Abcam, UK), donkey anti-mouse IgG H&L (Alexa Fluor 594) (ab15010B, 1:1000, Abcam, UK), and DAPI (Biosharp, Hefei, China) for 2 h under dark conditions. Images were acquired with a Leica SP8 (Wetzlar, Germany) confocal microscope.

HepG2 cells were cultured on coverslips in a 6-well plate. After exposure to vehicle control, DEX, EPI or NE for 4 or 48 h, the cells were washed with PBS and fixed with 4% formaldehyde in PBS at room temperature for 15 min. The fixed cells were washed with 0.2% Triton X-100 at 4°C for 2 min, incubated with ice-cold methanol at −20°C for 10 min. After rinsing with PBS at room temperature for 5 min, the cells were blocked with 10% blocking serum (Beijing Solarbio Science & Technology Co., Ltd.) in PBS at 37°C for 30 min. The cells were then probed with anti-γ-H2AX antibody (1:200 dilution) overnight at 4°C followed by washing with PBS containing 1% BSA and incubation with FITC-conjugated goat anti-rabbit IgG (1:50 dilution; cat. no. ZF-0311; ZSGB Biotech) at room temperature for 1 h. After washing thrice with 1% BSA (cat. no. AP0027; NOVON Scientific) in PBS, the coverslips were incubated with 4′,6-diamidine-2-phenylindole dihydrochloride (300 nM in PBS) for 30 min in the dark. The coverslips were then washed thrice with distilled water and mounted on slides before viewing with an Olympus DP72 microscope (Olympus Corp.).