T akt

DOX was purchased from Haizheng Pfizer Pharmaceutical Co., Ltd. Levosimendan was obtained from Orion Corporation, Espoo, Finland. The following primary antibodies were purchased from Cell Signaling Technology (Boston, MA, USA): Bcl-2-associated X protein (BAX; 1 : 1000), c-caspase-3 (1 : 1000), PTEN (1 : 1000), P-Akt (1 : 1000), T-Akt (1 : 1000), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1 : 1000), and B-cell lymphoma-2 (Bcl-2) (1 : 1000) was purchased from Abcam. Akt inhibitor (Akt i) was purchased from Sigma-Aldrich (St. Louis, MO, USA). A goat anti-rabbit secondary antibody was purchased from LI-COR Biosciences (Lincoln, USA). The BCA protein assay kit was obtained from Dōjindo Laboratories (Kumamoto, Japan).

Lung tissue homogenate was analyzed for TGF-β, t-AKT, p-AKT, GSH, and MDA protein levels using an enzyme-linked immunosorbent assay (ELISA) reader (Stat Fax 2200, Awareness Technologies, Palm City, FL, USA). Rat ELISA kits were used for the detection of TGF-β (Bio Vision incorporated, Cambridge, UK), t-AKT, p-AKT (Abcam, Cambridge, UK), GSH (Bio Vision incorporated, Cambridge, UK), and MDA (Bio Vision incorporated, Cambridge, UK).

Samples of frozen SC-WAT were homogenized in lysis buffer with 1 M HEPES, 2 M NaCl, 20% SDS, 0.5 M EDTA, 100mM Benzamidina (pH = 7.4), protease and phosphatase inhibitor cocktail EDTA-free (Thermo Scientific) at the concentration of 10 μl/ml. Samples were centrifuged for 15 min at 15.000 rpm at 4°C. Protein concentrations of the homogenates were measured by the BCA method with a protein assay kit (PIERCE Biotechnology, Rockford, IL, USA) using bovine serum albumin as a standard. Aliquots (60 μg) of protein were subjected to SDS-PAGE. The membranes were incubated overnight at 4°C with the following primary antibodies: p- AKT (1:1000), t-AKT (1:1000), GLUT-4 (1:1000), AT2 (1:1000), MAS (1.25:1000) (Abcam, Cambridge, USA), p-HSL (1:1000), t-HSL (1:1000), Perilipin (1:1000), ATGL (1:1000), AT1 (1:1000), (Cell Signaling, Beverly, MA) and beta-actin (Abcam, Cambridge, USA). The signal on the membrane was detected via the peroxidase reaction in the ECL solution using an Image Quant LAS 4000 mini system (GE Healthcare Life Sciences). Band intensities were quantified based on optical densitometry measurements using the Image J program (version 1.43 for Windows).

DPC12 cells were pre-treated with 4 and 8 µg/mL SCWEA for 3 h, and followed with 24 h co-incubation with 25 mM l-Glu. Cells were lysed by radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich, USA) which contains 2% phenylmethanesulfonyl fluoride (PMSF; Sigma-Aldrich, USA) and 1% protease inhibitor cocktail (Sigma-Aldrich, USA). Proteins were separated via 12% SDS-PAGE gel and transferred electrophoretically onto nitrocellulose membranes (Bio Basic, Amherst, NY, USA). The transferred membranes were then blotted with antibodies as follows: Bcl-2, Bcl-xL, T-AKT, P-AKT, T-GSK-3β, P-GSK-3β and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:1000; Abcam, Cambridge, UK) at 4 °C overnight, followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz, CA, USA). Chemiluminescence was detected by using ECL detection kits (GE Healthcare, Bucks, UK). The intensity of the bands was quantified by scanning densitometry using software Image J (National Institutes of Health, Bethesda, MD, USA).

Total protein was extracted using RIPA buffer containing protease and phosphatase inhibitors. The protein samples were heated at 100℃ for 10 min, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto PVDF membranes (EMD Millipore, Billerica, MA). The membranes were blocked in 5% bovine serum albumin (BSA) at room temperature for 1 h. Immunoblotting was performed with antibodies against β-actin, VEGFA, p-Akt, t-Akt, and HIF-1α (Abcam, Cambridge, MA). Then, the blot was detected with horseradish peroxidase-labeled secondary antibody. Signals were visualized using the ECL detection system (Millipore, Boston, MA) according to the manufacturer’s instructions.

The protein samples were extracted from the cells with RIPA buffer including the PMSF. The supernatant solution was collected after centrifuging at 8 000 rpm for 10 min. Same amounts of proteins were separated with SDS-PAGE. Then, the proteins were transferred to a PVDF membrane (Sigma, USA). After blocking with TBST, the membrane was incubated with anti-GLUC2, T-AKT, p-AKT, p-FOXO1, T-FOXO1, Pdx1, and β-actin (1 : 1500, Abcam, Beijing, China) overnight at 4°C. After washing twice, the membrane was incubated with secondary antibody (antimouse HRP-conjugated antibody, 1 : 2000) for 1 h. Finally, the protein was detected by chemiluminescence with Thermo ECL Substrate (Bio-Rad) and analyzed using the ImageJ software.