Oil red o powder

Dulbecco modified eagle medium (DMEM) medium and fetal bovine serum (FBS) were obtained from Gibco (Grand Island, NY, USA). Insulin, 3-isobutylmethylxanthine (IBMX), indomethacin, 3,3′,5-triiodo-L-thyronine (T3), dexamethasone, troglitazone, Oil-red O powder, and CL316,243 were obtained from Sigma Aldrich (St Louis, MO, USA). Poly-vinylidene difluoride (PVDF) was procured from Millipore (Merck KGaA, Darmstadt, Germany). The electrochemiluminescence (ECL) kit was obtained from GE Healthcare Life Sciences (Seoul, Korea).

Oil red O powder (Cat#O0625, Sigma-Aldrich, USA) was dissolved in 2-propanol (0.5%). The stock was then diluted to 0.3% oil red O solution with distilled H₂O and filtered through 0.22-μm filter. Fibroblast cells were seeded in 24-well plate (1E4/well) overnight. After treatment with 10 nM MPA for 24 h, the treated cells (CN 2,4 and CO 1,4) were washed with ice cold PBS and incubated with 60% isopropanol for 5 min. The coverslips were left air-dried before staining with Oil Red O dye for 15 min. Finally, the stained cells were washed three times with Millipore water, and counterstained with hematoxylin for 30 s and washed with PBS for three times. The cells were observed with a Nikon Eclipse E200 microscope (Nikon Instruments, Japan). Experiments were repeated twice and the representative data were shown. The stained lipid droplets were quantified using ImageJ software.

3-Isobutyl-1-methylxanthine (IBMX), dexamethasone (DEX), insulin, and Oil Red O powder were purchased from Sigma-Aldrich Co. LLC (St. Louis, MO, USA). Dulbecco's modified Eagle's medium (DMEM), bovine serum (BS), fetal bovine serum (FBS), and Antibiotic-Antimycotic (ABAM) were purchased from Life Technologies, Inc. (Grand Island, NY, USA). Orlistat was purchased from Tokyo Chemical Inc. (Tokyo, Japan). HFD (30%) was obtained from Research Diets (New Brunswick, NJ, USA). PPARγ, C/EBPα, SREBP1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) oligonucleotide primers were purchased from Bioneer Corporation (Daejeon, Republic of Korea), and SYBR Premix Ex Taq was purchased from Takara Bio Inc. (Otsu, Japan). Antibodies against PPARγ (cat. No. sc-7273), C/EBPα (cat. No. sc-9314), SREBP1 (cat. No. sc-13551), C/EBPβ (cat. No. sc-150), and β-actin (cat. No. sc-81178) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Horseradish peroxidase conjugated secondary antibodies were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, USA).

Rb1 (>98%, ab142646) was purchased from Abcam (Cambridge, UK). 3-Isobutyl-1-methylxanthine (IBMX), dexamethasone (Dex), insulin, and Oil Red O powder were purchased from Sigma (St. Louis, MO, United States). L748337 was from Tocris Bioscience (Bristol, UK). Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) were purchased from Corning (NY, United States). Antibodies for liver kinase B1 (LKB1) (3047S), pLKB1 (Ser428) (3482S), AMP-activated protein kinase alpha (AMPKα) (2532S), pAMPKα (Thr172) (2535S), acetyl-CoA carboxylase (ACC) (3676S), pACC (Ser79) (3661S), silent information regulator T1 (SIRT1) (8469S), SIRT3 (5490S), phospho-hormone sensitive lipase (pHSL) (Ser563) (4139S), phospho-PKA substrate (9624S), UCP1 (14670S), and β-actin (3700S) were purchased from Cell Signaling Technology (Beverly, MA, United States); the antibody for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (sc-32233) antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States); antibodies for PGC1α (ab54481), comparative gene identification 58 (CGI58) (ab59488), adipose triglyceride lipase (ATGL) (ab207799), HSL (ab45422), and β3AR (ab94506) were purchased from Abcam (Cambridge, UK); the antibody for PPARα (PA1-822A) was purchased from Thermo Fisher Scientific (Waltham, MA, USA).

Liver and skeletal muscle tissue from euthanized animals were snap frozen, cryosectioned (10 µm thick) and stained with Oil Red O for neutral lipid content as previously described [34] (link). Muscle and liver sections were fixed with 3.7% formaldehyde for 1 h at room temperature while an Oil Red O solution composed of 0.5 g Oil Red O powder (Sigma-Aldrich, Canada) and 100 ml of 60% triethyl phosphate (Sigma-Aldrich, Canada) was mixed and filtered. Following fixation in 3.7% formaldehyde, slides were immersed in filtered Oil Red O solution for 30 minutes at room temperature. Slides immediately underwent five washes with ddH2O, were allowed to dry for 10 minutes and were sealed with Permount (Sigma-Aldrich, Canada). Skeletal muscle and liver images were acquired at 10× and 20× magnifications respectively using a Nikon Eclipse 90i microscope (Nikon, Canada) and Q-imaging MicroPublisher 3.3 RTV camera with Q-capture Software. Intensity of Oil Red O staining of IMCL droplets on serial sections of the tibialis anterior was assessed with Adobe Photoshop CS6, converted to greyscale and reported as the average optical density (60 fibers were counted per muscle section). The greyscale is evaluated on a range of 0 (completely black) to 255 (completely white).