Anti c ebpα

3-isobutyl-1-methylxanthine (IBMX), compound C (CC), dexamethasone (Dex), indomethacin, insulin, and Oil Red O powder were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). Bovine serum (BS), Dulbecco’s modified Eagle’s medium (DMEM), penicillin/streptomycin/glutamine (P/S/G) and were obtained from Gibco (Grand Island, NY, USA). Fetal bovine serum (FBS) was purchased from HyClone Laboratories Inc. (Logan, UT, USA).
Anti-C/EBPα, anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), anti-PPARγ coactivator 1 alpha (PGC1α) and anti-UCP1 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), and antibodies for AMPKα, pAMPKα, acetyl-CoA carboxylase (ACC), pACC, liver kinase B1 (LKB1), pLKB1 and PPARγ were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA).

FLAG-tagged HMMR expression vector was constructed by inserting PCR amplified HMMR fragment into the pcDNA3 vector (Invitrogen) linked with FLAG tag at the amino terminus. The HMMR promoter luciferase reporters were made by inserting PCR-amplified HMMR promoter fragments into the pGL4-Basic vector (Promega). HepG2 and MHCC-97H liver cancer cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA). Small interfering RNAs (siRNAs) were synthesized by JTS scientific or GemmaPharma. The cDNA target sequences of siRNAs and/or short hairpin RNAs (shRNAs) for HMMR and CEBPα were listed in Table S1. Stable cell lines overexpressing HMMR shRNA were established by lentiviral transduction using pSIH-H1-Puro carrying HMMR shRNA. Anti-cyclin D1, anti-cyclin E, anti-cyclin B1 and anti-CEBPα were purchased from Santa Cruz Biotechnology; Anti-HMMR was from Proteintech.

Control shRNA plasmid (Cat. No. sc-108060) and β3-AR shRNA plasmid (Cat. No. sc-39869) were purchased from Santa Cruz Biotechnology (Delaware, CA, USA). PD98059, SB203580 (Cat. No. BML-E1294) was purchased from Enzo (Farmingdale, NY, USA). SP600125 (Cat. No. 420119) was from Calbiochem (Madison, WI, USA). LY294002 (Cat. No. ST-420) and GF109203X (Cat. No. EI-246) were purchased from Biomol (Hamburg, Germany). 3-isobutyl-1-methylxanthine (IBMX), dexamethasone, and insulin were bought from Sigma (St. Louis, MO, USA). Primary antibodies specific for anti-β3-AR, anti-C/EBP-α, and anti-PPAR-γ, anti-actin, anti-phospho (p)-STAT-3 (Y705), anti-STAT-3, anti-phospho (p)-STAT-5 (Y694), and anti-STAT-5 were purchased from Santa Cruz Biotechnology (Delaware, CA, USA). Anti-perilipin A antibody was purchased from BioVision (Milpitas, CA, USA). The anti-FASN antibody was purchased from BD Bioscience (San Jose, CA, USA). Super Signal™ West Pico PLUS Enhanced chemiluminescence (ECL) (Cat. No. 34080) was purchased from Thermo Scientific (Waltham, MA, USA). All Plasticware, including 6-well, 24-well plates, 60 mm, and 100 mm cell culture dishes, were obtained from SPL Life Sciences (Pocheon-si, Korea).

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).

Tissues were homogenized with a Polytron homogenizer in an IntactProein extraction buffer (GenuIn Biotech, Blacksburg, VA, USA, catalog #415). After placing on ice for 15 min, the lysates were sonicated for 10 min, and debris was removed by centrifugation at 14,000 rpm for 10 min at 4 °C. The total protein was resolved on 8% or 10% SDS-PAGE after BCA determination of the protein concentration. The Western blotting signals were detected with ECL (Thermo Fisher Scientific, Waltham, MA, USA, catalog PI32209). The following antibodies were used in this study: anti-HSP90 (Proteintech, Rosemont, IL, USA, catalog # 13171-1-AP), anti-Adipsin (R&D systems, Minneapolis, MN, USA, catalog # AF5430), anti-Adiponectin (Invitrogen, Waltham, MA, USA, catalog # PA1-054), anti-PPARγ (Cell Signaling Technology, Danvers, MA, USA, catalog # 2443), anti-SirT1, anti-UCP1 (Abcam, Cambridge, UK, ab234430), anti-PGC-1α (Abcam, Cambridge, UK, catalog # ab54481), anti-C/EBPα (Santa Cruz, CA, USA, catalog # sc-61). Western blots were quantified using densitometry via Image J.