Tissue and Cell Lysis Protocol

To prepare tissue lysates, snap-frozen adipose tissues were weighed and homogenized with a Bullet Blender (Next Advance, Averill Park, NY, USA) in PLC lysis buffer supplemented with protease inhibitor cocktail (Roche), 1 mM PMSF, 10 μM TSA (Trichostatin A, Selleckchem, Houston, TX, USA) and 5 mM Nicotinamide (Alfa Aesar, Ward Hill, MA, USA).^{15 (link), 52 (link)} For cell lysates, the 3T3L1 adipocytes were washed with ice-cold PBS and homogenized with a Bullet Blender. Total protein concentrations of the lysates were determined using the DC protein assay (Bio-Rad). Western blotting and image analysis were conducted as described previously.^{15 (link)} Antibody catalog numbers and vendors are as follows: cleaved caspase-3 Rabbit mAb (9664) and LC3B antibody (no. 2775) from Cell Signaling Technology (Beverly, MA, USA); PPAR-gamma antibody (MA5-14889) and GAPDH antibody (MA5-15738) from Pierce (Rockford, IL, USA) or Thermo Fisher Scientific (Waltham, MA, USA); and HO1 antibody (3391-100) from Biovision (Milpitas, CA, USA).