Pparγ

Metformin (MET), simvastatin (SIM), isobutyl-3-methylxanthine (IBMX), dexamethasone (DEX), and insulin (INS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Sigma-Aldrich (St. Louis, MO, USA), and Penicillin/Streptomycin (P/S) was obtained from Capricorn Scientific GmbH (Ebsdorfergrund, Hessen, Germany). Primary antibodies against SREBP-1C and STAT3 were obtained from Abcam (Cambridge, UK), a primary antibody against SMAD3 was obtained from NSJ Bioreagents (San Diego, CA, USA), against PPARγ from Sigma-Aldrich (St. Louis, MO, USA), and C/EBPα from Antibodies-online.com (Ebsdorfergrund, Hessen, Germany). HRP-conjugated GAPDH Monoclonal antibody was obtained from Proteintech (Rosemont, IL, USA). Secondary anti-rabbit antibody labeled with HRP and the biotinylated goat anti-rabbit IgG (H+L) secondary antibody were obtained from Jackson ImmunoResearch (West Grove, PA, USA). BD anti-Streptavidin, PE-Cy™5 was purchased from Fisher Scientific (Hampton, NH, USA).

Cells were washed with PBS and proteins extracted in 50 μl of lysis buffer (50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 10% glycerol, and 1% NP-40) supplemented with 10 mM NaF, 1 mM Na₃VO₄, 10 μg/ml leupeptine, 1 μg/ml pepstatin and aprotinin, and 1 μl/ml PMSF saturated. Thirty micrograms of proteins was boiled at 95 °C in Laemmli buffer and electrophoresed in 10% SDS/PAGE gels. Separated proteins were transferred to PVDF membranes (20 V for 30 min) and blocked in TBS solution containing 0.1% Tween 20 and 5% non-fat dry milk for 1 h at room temperature. Immunodetection of specific proteins was carried out by incubation with primary antibody against HIF-1α (1:1000; BD Biosciences, #610959 San Jose, CA, USA), HIF-2α (1:1000; Novus Biologicals, Littleton, USA), PHD1 (1:1000; Abcam, Cambridge, UK), PHD2 (1:1000; Abcam), PHD3 (1:1000; Abcam), OH-HIF-1α (1:1000; Cell Signaling, Danvers, MA, USA), PPARγ (1:1000), β-actin (1:10.000; Sigma), and arginase 1 (N20) (1:500; Santa Cruz, Dallas, TX, USA) overnight at 4 °C. After washing membranes, horseradish peroxidase-conjugated secondary antibody was added and detected by chemiluminescence system (GE Healthcare Europe GmbH).

Cells (5000 cells/μl) were lysed in 3x Laemmli-buffer. To disrupt all cells, the suspension was heated for 10 min at 95°C followed by centrifugation at 8,000 x g.
10 μl (~20 μg) of the cell lysate were loaded on 10%-SDS-PAGE and separated. Transfer to a nitrocellulose membrane was performed with semi-dry blot (Biometra, DE) at 200 mA for 20 min. We used primary antibodies for SIRT1 (Merck Millipore, USA), SIRT3 (Merck Millipore, USA), SIRT4 (Thermo Scientific, DE), AMPKα1 (Merck Millipore, USA), PPARγ (Merck Millipore, USA), PGC1α (Merck Millipore, USA) and β-Actin (Cell Signalling, DE) as a marker. For detection, we used the Odyssey FC system (LI-COR Bioscience, USA) with appropriate secondary antibodies.