Anti hnf4α

PLC/PRF/5, human hepatoma cell line was obtained from the American Type Culture Collection (Rockville, MD). The cells were cultured in low glucose (5.5 mM) or high glucose (25 mM) Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS; Gibco) and antibiotic. Mito Tracker Green and Mito Tracker red CMXRos were obtained from Invitrogen. Sodium lactate and sodium dichloroacetate were obtained from Sigma-Aldrich. The antibodies obtained were: anti-CD133/1 (AC133)-PE and anti-CD133 (W6B3C1) pure (Miltenyi Biotec), anti-HNF4α (Santa Cruz Biotechnology), anti-HNF1α and anti-HNF3α (Millipore), anti-HNF1β (Genway), anti-PDK4 (ABGENT), and anti-β-actin (Sigma). Synthetic siRNAs against LDHA and PDK4, miR-122, U6 primers and mimic were obtained from Qiagen. Cells were transfected at a final concentration of 50 nM siRNA or 20 nM of miR-122 mimic using Oligofectamine reagent (Invitrogen) according to the manufacturer's instructions. The lentiviral particle expressing miR-122 and lentiviral negative control mimic were obtained from GenCopoiea.

For intracellular staining of ALB and AAT, the cells were fixed with 4 % paraformaldehyde for 15 minutes at room temperature and then incubated with PBS containing 0.2 % Triton X-100 (Sigma) for 15 minutes. Cells were then washed three times with PBS. After being blocked by 3 % bovine serum albumin (BSA) in PBS for 60 minutes at room temperature, cells were incubated with primary antibodies at 4 °C overnight, washed three times with PBS, and then incubated with appropriate fluorescence-conjugated secondary antibody for 60 minutes at 37 °C in the dark. Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI; Sigma). Primary and secondary antibodies were diluted in PBS containing 3 % BSA. Antibodies used for immunofluorescence are as follows: sheep anti-ALB (1:500; Bethyl), rabbit anti-AAT (1:1; Abcam, Chengdu, Sichuan, P.R. China), rabbit anti-hepatocyte nuclear factor alpha-4 (anti-HNF4α, 1:50; Santa Cruz), rabbit anti-Ki67 (1:100; Santa Cruz), dylight 488 conjugated donkey anti-rabbit IgG (1:200; Bethyl), dylight 594 conjugated donkey anti-sheep IgG (1:200; Bethyl), and Alexa Fluor 594 conjugated goat anti-rabbit IgG (1:100; ZSBG-BIO, Chengdu, Sichuan, P.R. China).

The harvested cells and obtained tissues were lysed by RIPA buffer [20 mM Tris/HCl, 1% NP-40, 0.5% phosphatase inhibitor cocktail (Sigma, St Luis, MO, USA), 150 mM NaCl, 2 mM KCl, pH 7.4]. The debris was removed by centrifuging at 12000 RPM for 20 min. After the filtering of cell debris, levels of protein in the lysate were quantified by a BCA protein assay kit (Thermo Fisher scientific, Waltham, MA, USA). The samples were mixed with 5X SDS sample buffer and boiled at 95 °C for 4 min. The proteins were separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membrane (Millipore, Burlington, MA, USA). The transferred membranes were washed with TBS-T. Antibodies were used in 1:2000 ratios. The primary antibodies used were as follows: anti-HNF4α (Santa Cruz, CA, USA), anti-HNF1α (Santa Cruz), anti-HNF3β (Santa Cruz), anti-C/EBPα (Santa Cruz), anti-HBcAg (Dako, Glostrup, Denmark), anti-HBx protein (Abcam, UK), anti-p-ERK (Cell Signaling Technology, MA), anti-ERK (Cell Signaling Technology), anti-p-AKT (Cell Signaling Technology), anti-AKT (Cell Signaling Technology), anti-p-JNK (Cell Signaling Technology), anti-JNK (Cell Signaling Technology), anti-p-p38 (Cell Signaling Technology), anti-p38 (Cell Signaling Technology), anti-β-actin (Sigma-Aldrich Co, St Louis, MO, USA).