Bicinchoninic acid method

Total cellular proteins were extracted by lysing cells with buffer containing 150 mM NaCl, 0.1% Triton X-100, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 50 mM Tris-HCl (pH 7.0) and 1 mM ethylenediaminetetraacetic acid. Protein concentrations were determined using the bicinchoninic acid method (Beyotime Institute of Biotechnology, Haimen, China). MC protein extracts (50 µg/lane) were separated by 10% SDS-polyacrylamide gel electrophoresis and were transferred to nitrocellulose membranes. Following blocking with 5% skimmed milk in Tris-buffered saline (pH 7.6) at room temperature for 1 h, the membranes were incubated at 4°C overnight with primary antibodies against LC3 (1:1,000), p62 (1:1,000), Atg7 (1:1,000) and β-actin (1:100). Membranes were then incubated with Alexa Fluor 790-conjugated goat anti-rabbit (1:2,500; catalog no. A27041; Thermo Fisher Scientific, Inc.) or Alexa Fluor 680-conjugated goat anti-mouse (1:5,000; catalog no. A28183; Thermo Fisher Scientific, Inc.) secondary antibodies, at room temperature for 1 h. Protein bands were visualized with the LI-COR Odyssey^® protein analysis system (LI-COR Biosciences, Lincoln, NE, USA). The relative intensity of each band was normalized to β-actin using Image J software version 1.48 (National Institutes of Health, Bethesda, MD, USA).

The total amount of protein was detected by the bicinchoninic acid method (Beyotime, Shanghai, China). Proteins (20 μg) were separated by SDS-PAGE on 10% polyacrylamide gels and transferred electrophoretically to polyvinylidene fluoride membranes with the TGX Stain-Free FastCast Acrylamide Kit (Bio-Rad, California, USA). The membranes were blocked with 5% nonfat dry milk in TBS + 0.1% Tween-20 for 1 h and incubated overnight at 4 °C with the primary antibodies in bond primary antibody diluent (see Supplementary Table 2, Supplemental digital content 1, http://links.lww.com/WNR/A654, for antibody information), followed by secondary antibodies [horseradish peroxidase (HRP)–labeled goat antimouse immunoglobulin (Ig)G, 1:5000, and HRP-labeled goat antirabbit IgG, 1:10 000, Abcam, Cambridge, UK] at room temperature for 1 h. The proteins were detected by the Bio-Rad ChemiDoc Touch Image System (Bio-Rad).

The ICP2 cells were seeded at 5 × 10⁴ in a 24-well plate and infected with Lenti-PPARγ1, Lenti-PPARγ2, and Lenti-control viruses at 50 MOI with or without rosiglitazone (20 μmol/L). For adipocyte differentiation, ICP2 cells at about 60% confluence were incubated with the differentiation medium containing Dulbecco's Modified Eagle Medium/Ham's F-12 culture medium, 10% fetal bovine serum, 1% antibiotic–antimycotic solution, and 160 μmol/L sodium oleate (Sigma). The differentiation medium was changed every day until the indicated time points.
Differentiated ICP2 adipocytes were washed twice with PBS, fixed in 4% formaldehyde for 15 min at room temperature, and rinsed 3 times with distilled water. The cells were stained with Oil red O solution (3:2, 0.6% Oil Red O in isopropanol:water) for 15 min at room temperature, then washed with distilled water. The intracellular lipid droplets were visualized with an optical microscope (Leica). To quantify staining, Oil Red O was extracted from the cells with isopropanol solution, and the optical density was measured at a wavelength of 510 nm. The cell total protein concentration was estimated by the bicinchoninic acid method (Beyotime, China). Intracellular lipid content was normalized against the protein to allow an accurate comparison.