Protein lysis buffer

Cells were washed with ice-cold PBS and extracted in a protein lysis buffer (Intron Biotechnology, Seoul, Korea). Protein concentrations were determined by the Bradford assay. The cell lysates were mixed with 5 Χ SDS sample buffer, boiled for 4 min, and then separated on 10% SDS-PAGE. After electrophoresis, proteins were transferred to polyvinylidene difluoride membranes. The membranes were blocked in 2% bovine serum albumin (BSA) for 30 min, washed, and incubated overnight at 4 °C with specific primary antibodies in Tris-buffered saline 2% BSA and 0.1% Tween-20 (TBS-T). The membranes were washed three times to remove the primary antibodies and incubated for 2 h with a horseradish peroxidase-conjugated secondary antibody (1:1000–2000). After washing three times with TBS-T, immuno-positive bands were visualized using the ECL chemiluminescent system (Amersham Pharmacia Biotech, ON, Canada) and analyzed using ImageQuant Las-4000 (GE Healthcare Life Science, WI, USA).

Cells were washed with ice-cold PBS and extracted in protein lysis buffer (Intron Biotechnology, Seoul, Korea). The total protein content of the samples was determined according to the Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA) using a bovine serum albumin (BSA) standard curve. Protein samples of cell lysate were mixed within equal volume of SDS sample buffer, boiled for 5 min and were loaded on 10–12% SDS-PAGE gels. After acrylamide gel-electrophoresis, proteins were transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked in 5% non-fat dry milk for 30 min, washed and were incubated for overnight at 4 °C with specific primary antibodies against cyclin E, total Akt, phospho-Akt, and β-actin in Tris-buffered saline (TBS) containing Tween-20 (0.1%). Primary antibodies were removed by washing the membranes three times in TBS-T and the membranes were incubated for 2 h with horseradish peroxidase-conjugated secondary antibody (1:1000–2000). Following the washing procedures in TBS-T, immune-positive bands were visualized by enhanced chemiluminescene and exposed to ImageQuant LAS-4000 (Fujifilm Life Science, Tokyo, Japan). The relative intensities of protein bands were determined by NIH Image 1.59 software (Image J) (NIH, Bethesda, MD, USA) and then all values were subsequently normalized to untreated group.

D3G (chemical formula C₂₁H₂₁O₁₂Cl, >97% purity) was purchased from Polyphenols AS (Sandnes, Norway). Dorsomorphin, AICAR, indomethacin, 3-isobutyl-1-methylxanthine (IBMX), and dexamethasone (DEX) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), bovine calf serum (BCS), trypsin-EDTA, and fetal bovine serum (FBS) were procured from Thermo Fisher (San Jose, CA, USA). CCK-8 was purchased from Dojindo Molecular Technologies (Rockville, MD, USA). Insulin was purchased from Thermo Fisher (San Jose, CA, USA). Random hexamers and the reverse transcriptase enzyme mix (GoScriptTM) were obtained from Promega (Madison, WI, USA). Monoclonal antibodies against C/EBPα, SREBP1, PPARγ, FAS, ACC, SIRT1, p-ACC, AMPK, p-AMPK, CPT-1, and horseradish peroxidase (HRP)-coupled anti-rabbit or anti-mouse secondary antibodies were purchased from Abcam (Cambridge, UK). Total RNA was extracted using an easy-spin^TM Total RNA Extraction Kit (iNtRON Biotechnology, Seongnam-si, Korea) and a PRO-MEASURE^TM protein measurement solution, protein lysis buffer, and a Western blot detection system were purchased from iNtRON Biotechnology (Seongnam-si, Korea). The polyvinylidene difluoride (PVDF) membrane was procured from Merck (Burlington, Massachusetts, USA).