C ebpα

Protein lysates were stored in 1× SDS and separated by SDS-PAGE. The nitrocellulose membranes transferred with proteins were blocked in 5 % BSA, incubated overnight at 4 °C with specific primary antibodies, washed and incubated for 1 h with the corresponding secondary antibodies. A Tanon 5200 automated chemiluminescence imaging analysis system was subsequently implemented. The following primary antibodies were used in this study: tubulin (2146 S; Cell Signaling Technology, Danvers, USA; 1:1000), C/EBP-α (2295 S; Cell Signaling Technology, Danvers, USA; 1:1000), PPAR-γ (2435T; Cell Signaling Technology, Danvers, USA; 1:1000), C/EBP-β (3082 S; Cell Signaling Technology, Danvers, USA; 1:1000), LPL (sc-373; Santa Cruz Biotechnology, Dallas, USA; 1:800), FABP4 (2120 S; Cell Signaling Technology, Danvers, USA; 1:1000), and IRS-1 (ab52167; Abcam, Cambridge, UK; 1:1000).

d-glucose, 3-isobutyl-1-methylxanthine, dexamethasone, insulin, ethanol (EtOH), Oil-Red O, propylene glycol, dimethyl sulfoxide (DMSO), pioglitazone, metformin, dorsomorphin (an AMPK inhibitor, AI), and all other chemicals were of analytical grade and obtained from Sigma-Aldrich Inc. (St. Louis, MO, USA). Normal glucose (100 mg/dL) Dulbecco’s Modified Eagle Medium (DMEM; Catalog No.: 12320), penicillin/streptomycin (P/S), fetal bovine serum (FBS), horse serum, and trypsin-EDTA were bought from Invitrogen (Carlsbad, CA, USA). Rosiglitazone used in this experiment did not contain any inactive ingredients, and the pure compound was kindly provided by GlaxoSmithKline, Ltd. (Taipei, Taiwan). Phospho-AMPK, AMPK, phospho-ACC, ACC, phospho-AKT, AKT, PPARγ, C/EBPα, C/EBPβ, and GAPDH antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Proteins were extracted from cell monolayers as described previously (Gorwood et al., 2019 (link); Gorwood et al., 2020 (link)). After SDS-PAGE, the proteins were transferred to nitrocellulose membranes. Specific proteins were detected using antibodies against p16^INK4A, p21^WAF1 (BD Bioscience, Franklin Lakes, NJ), prelamin A, PPARγ, CEBPα, SREBP1c, AMPK, phospho-AMPK (Cell Signaling Technology, Danvers, MA), and the protein loading control tubulin (Merck, Sigma-Aldrich). Immunoreactive complexes were detected using HRP-conjugated secondary antibodies (Cell Signaling Technology, Danvers, MA) and enhanced chemiluminescence (Thermo Fisher Scientific).

Liver tissues from each mouse were homogenized at 4 °C in extraction buffer (100 mM Tris-HCl, pH 7.4, 5 mM EDTA, 50 mM NaCl, 50 mM sodium pyrophosphate, 50 mM NaF, 100 mM orthovanadate, 1% Triton X-100, 1 mM phenylmethanosulfonylfluoride, 2 mg/mL aprotinin, 1 mg/mL pepstatin A, and 1 mg/mL leupeptin) and centrifuged at 3000×g for 15 min at 4 °C. HepG2-HBx cells were lysed in 250 μL of sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 50 mM dithiothreitol, and 0.1% bromophenol blue). The amount of proteins in the tissue lysate and cell lytsate were assayed using the Bradford assay. Proteins (40 μg/sample for homogenized tissue, 10 μg/sample for cell lysates) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and protein bands were transferred electrophoretically to nitrocellulose membranes. Membranes were probed with polyclonal antibodies against SREBP1 (Santa Cruz Biotechnology, Santa Cruz, CA), GFP, AKT, AKT-p, PI3K, PI3K-p, C/EBPα, and β-actin (as a loading control) (Cell Signaling Technology, Beverly, MA, USA). Bound antibodies were detected using peroxidase-conjugated anti-rabbit antibodies followed by chemiluminescence assay (ECL System; Amersham, Buckinghamshire, UK) and autoradiographic exposure. The intensity of band on the bolts was calculated by Gel-Pro® Analyzer (Media Cybernetics, Inc., Silver Spring, MD).

All reagents used in the experiment were guaranteed reagent grade and HPLC-grade. Acetonitrile, ethanol and methanol were from Merck (Darmstadt, Germany). Isopropanol (100%) was from J.T. Baker Chemical (Phillipsburg, NJ, USA). Phosphate-buffered saline (PBS) was from Lonza (Walkersville, MD, USA). Dulbecco’s modified Eagle’s medium (DMEM) was from BioWest (Riverside, MO, USA). Paraformaldehyde (4%) was from Biosesang (Seongnam-si, Korea). Bovine calf serum (BCS), fetal bovine serum (FBS), penicillin/streptomycin (P/S) and insulin were from Gibco (Grand Island, NY, USA). Ginsenoside Rg₁, Rb₁ and Rg₃(S) were from ChromaDex Co. (Irvine, CA, USA). Dexamethasone (Dex), 3-isobutyl-1-methylxanthine (IBMX), thiazolyl blue tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), Oil Red O, glycyrrhizin, 2-methyl-2-butanol and 2,2,2-tribromoethanol (Avertin) were from Sigma-Aldrich (St. Louis, MO, USA). RIPA buffer and bicinchoninic acid (BCA) protein assay kit were from Thermo Fisher Scientific (Waltham, MA, USA). Primary antibodies, anti-rabbit β-actin, PPARγ, C/EBPα, adiponectin, AMPK, p-AMPK, ACC, p-ACC were from Cell Signaling Technology (Danvers, MA, USA), SREBP-1c and CPT-1 from Santa Cruz Biotechnology (Dallas, TX, USA).

To determine the effect of bavachin on protein expression of PPARγ, C/EBPα and GLUT4, cells were lysed at D5. To detect Akt and AMPK activation and GLUT4 translocation, 3T3-L1 pre-adipocytes were pre-incubated for 1 h with bavachin followed by insulin stimulation for another 1 h. Total protein was electrophoresed in SDS-polyacrylamide gels and transferred to polyvinylfluoride membranes. The membrane was probed with primary antibodies against PPARγ, C/EBPα, phospho-Akt, phospho-AMPK, or GLUT4 (Cell Signaling Technology, Danvers, MA, USA). The plasma membrane fraction was prepared using a modified ultracentrifugation method [35 (link)].

Rabbit antibodies for GEF-H1, myosin phosphatase targeting protein 1 (MYPT1), phospho-MYPT1 (Thr696), Akt, phospho-Akt (Ser473), FoxO1, phospho-FoxO1, PPARγ, and C/EBPα were obtained from Cell Signaling Technology, Inc. (Danvers, MA). Rabbit polyclonal anti-GEF-H1 antibody was also purchased from Abcam (Cambridge, UK) and used for immunostaining. Mouse monoclonal anti-tubulin (clone TUB 2.1) and anti-actin (clone AC-40) antibodies and sucralose were obtained from Sigma (St. Louis, MO). Sodium saccharin, cholera toxin and Y-27632 were from Wako Pure Chemical Industries (Osaka, Japan).