Prdm16

Curcumin (the chemical structure is shown in figure 1) with a purity of 98% was purchased from Sigma (cas.#: 458-37-7, St. Louis, MO, USA). Dulbecco's Modified Eagle's Medium (DMEM), penicillin-streptomycin, together with 0.25% trypsin-EDTA were obtained from Gibco (Rochester, NY, USA). Fetal bovine serum (FBS) was purchased from Tian Hang Bio-company (Hangzhou, China). Insulin, dexamethasone (DEX) and 3-isobutyl-1-methylxanthine (IBMX) were bought from Sigma. Oil Red O was obtained from Solarbio Biocompany (Beijing, China). The XF Cell Mito Stress Test kit was purchased from Seahorse Bioscience (Billericay, MA, USA). GW9662 was purchased from Selleck (cat.# S2915, Shanghai, China). Antibodies for uncoupling protein 1 (UCP1), PPARγ, peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) and GAPDH were purchased from Protein Tech Group (cat.# 23673-1-AP, 20658-1-AP, 16643-1-AP and 10494-1-AP, Rosemont, IL, USA). The antibody against PR domain protein 16 (PRDM16) was bought from Abcam, Inc. (cat.# ab106410, Cambridge, MA, USA). Anti-rabbit lgG-HRP was purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Unless otherwise specified, all other reagents were obtained from Beijing Sinopharm Chemical Group (Beijing, China).
Figure 1.

Chemical structure of Curcumin.

Total proteins were extracted using a mammalian protein extraction reagent (Thermo Fisher Scientific, Waltham, MA, USA) containing a protease inhibitor (Sigma-Aldrich, USA), and the protein concentrations were measured using a Bradford assay (Bio-Rad Laboratories, Richmond, California). Equal amounts of total protein (30 μg) were loaded onto 7.5% SDS-PAGE gels (Bio-Rad Laboratories, Richmond, California) at 200 V for 40 min, electro transferred to PVDF membranes, blocked in 5% non-fat milk at room temperature for 1 h, and incubated with the diluted primary UCP1 (Santa Cruz: sc-293418), PRDM16 (Abcam: ab106410), PGC1α (Santa Cruz: sc-518025), and β-actin (Santa Cruz: sc-47778) antibodies at 4 °C overnight in turn. After washing three times with PBST, the membranes were probed with horseradish-peroxidase-conjugated secondary antibody at room temperature for 1 h. β-actin was used as the loading control. The protein was detected using Pierce ECL Western blotting substrate (Thermo Fisher Scientific, Waltham, MA, USA), and the protein band density was quantified using ImageQuant TL (version 8.1; Amersham, Amersham, UK).

To obtain tissue lysates, eWAT and sWAT were homogenized in 10% sodium dodecyl sulfate buffer containing protease and phosphatase inhibitors. An antibody targeting PR domain containing 16 (PRDM16) (Abcam) was used for western blotting. Bands were detected with ECL enhanced chemiluminescence detection reagents (Applygen, Beijing, China) and scanned with an Alpha Imager 5500 (Alpha Innotech, San Leandro, CA, USA) imaging densitometer.

Cells were lysed in Pro-Prep solution (iNtRON Biotechnology, Seoul, Korea) containing phosphatase and protease inhibitors (Sigma-Aldrich, St. Louis, MO, USA). The lysates were clarified by centrifugation at 13,000× g for 150 min at 4 °C, and their protein concentrations were measured using a Bradford Assay (Bio-Rad, Hercules, CA, USA). The proteins in each sample (20 µg) were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes. The membranes were blocked using 5% non-fat dried milk solution, and then immunoblotted with primary antibodies (1:1000) targeting carnitine palmitoyl transferase (CPT)1, PGC1α, PRDM16, UCP1 (all from Abcam, Cambridge, UK), C/EBPα, PPARγ, FABP4, ATGL, AMP-activated protein kinase (AMPK), p-AMPK (all from Cell Signaling Technology, Danvers, MA, USA), DGAT1, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA). The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (Bio-Rad, Hercules, CA, USA) for 6 h (1:2000), and the reactive bands were detected using LAS image software (Fuji, New York, NY, USA).

CL316243, isoproterenol, resveratrol, rosiglitazone, amlexanox, zardaverine, H89, IBMX, and thiazolyl blue tetrazolium blue (MTT) were from Sigma. [1-¹⁴C] palmitic acid was from Moravek Biochemicals. 2′3′-cGAMP and 2′3′-cGAMP control were from Invivogen. ABT-737, Q-VD-OPh and nigericin were from Cayman Chemical. Digitonin was from EMD Millipore. Antibodies to UCP1 (Cat# Ab23841), C/EBPβ (Cat# Ab32358), ChREBP (Cat #157153), Prdm16 (Cat# ab106410), and ERP57 (Cat# Ab10287) were obtained from Abcam; antibodies to cGAS (Cat# 31659), p-TBK1 (Cat# 5483), TBK1 (Cat# 3013), p-IRF3 (Cat# 4947), IRF3 (Cat# 4302), STING (Cat# 13647), PKA Substrates phosphorylation (Cat# 9621), p-HSL (Cat# 4139), HSL (Cat# 4107), PPARγ (Cat# 2443), ATGL (Cat# 2439), FASN (Cat# 3180), p-ACC (Cat# 3661), ACC (Cat# 3662), and SCD1 (Cat# 2794) were all from Cell Signaling; an antibody to PGC1α (Cat# ST1204) was from EMD Millipore; an antibody to TNFα (Cat# CG1601) was from Cell Applications, Inc; an antibody to Complex IV (Cat# A6403) was from Molecular Probes; Lamin A antibody (Cat# 3267-100) was from BioVision; antibodies to beta-tubulin and myc were made from monoclonal antibody-producing cell lines obtained from ATCC; A polyclonal anti-DsbA-L antibody is homemade from rabbit and also available in EMD Millipore (Cat# ABS1644).

Cells were lysed in RIPA buffer for 40 min at 4 °C centrifuge. Removing insoluble material by centrifugation at 12,000× g for 15 min at 4 °C, and the supernatants were used to assay protein levels. Protein samples (50 μg) were separated by electrophoresis on 12% and 5% SDS-PAGE gels using slab gel apparatus and then transferred to PVDF nitrocellulose membranes (Millipore, Boston, MA, USA) blocked with 5% Skim Milk Powder/Tween 20/TBST at room temperature for 2 h. Primer antibodies against Mark4 and GAPDH were purchased from Bioworld (MN, USA). Antibodies against LC3B-ll and LC3l, P62, UCP1, PGC1α, Prdm16, and Cidea were from Abcam (Cambridge, MA, USA). Additionally, antibodies against AMPK, phospho-AMPK, AKT, phospho-AKT, mTOR, and phospho-mTOR were from Cell Signaling (Danvers, MA, USA). Membranes were incubated with primary antibodies at 4 °C overnight and then incubated with the appropriate HRP-conjugated secondary antibodies (Boaoshen, China) for 2 h at room temperature. Proteins were visualized using chemiluminescent peroxidase substrate (Millipore, Boston, MA, USA), and then the blots were quantified using ChemiDoc XRS system (Bio-Rad, Shanghai, China) and quantitative analysis of immune-blotted bands was performed using Quality One software (Bio-Rad).