Protease and phosphate inhibitors

Total cell lysates from all protein samples in this manuscript were incubated on ice in lysis buffer for 20 min (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM EDTA, 1× protease and phosphate inhibitors (Sigma-Aldrich)) followed by three cycles of 15 s sonication (Bioruptor). Lysates were subjected to Western blot analysis to detect Ty tag (1:500, SAB4800032 Sigma-Aldrich), Flag tag (1:5000, Sigma-Aldrich, clone M2, F1804), β-actin (1:10,000, C4, Santa Cruz Biotechnology, sc47778), HA tag (1:1000, Sigma-Aldrich, 3F10, 000000011867423001), H3 (1:10.000, Abcam, ab4729), PPARβ (1:500, Santa Cruz Biotechnology, F-10, sc-74517), total oxphos (1:2000, Antibody Cocktail, Abcam, ab110413), AMPKα (1:2000, Cell Signaling Technology, D5A2, 5831), and phospho-AMPKα T172 (1:2000, Cell Signaling Technology, 40H9, 2535). For detection of LC3B (1:2000, Cell Signaling Technology, 2775) the cells were additionally subjected to a 24 h treatment with chloroquine (Sigma-Aldrich, ref. C6628; 25 μM) before lysis. Western blots were either exposed on films or scanned with the BioRad ChemiDoc Touch Imaging System. Uncropped western blots can be found in Supplementary Fig. 8.

hWJ-MSCs were lysed using Mammalian Protein Extraction Reagent (M-PER, Thermo Fisher Scientific, San Diego, CA, USA) containing protease and phosphate inhibitors (Sigma-Aldrich Co., St. Louis, MO, USA). Protein concentrations were measured using Bradford Reagent (Sigma-Aldrich Co., St. Louis, MO, USA). Cellular lysates were resuspended in 4× Laemmli Sample Buffer and incubated at 95 °C for 5 min. Twenty micrograms of cellular lysates were separated to SDS-PAGE on a 10% Mini-PROTEAN^® TGX Stain-Free™ Precast Protein Gels (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and transferred to a 0.2 µm nitrocellulose membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA) using the Trans-Blot^® Turbo™ Transfer System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). After blocking, nitrocellulose membrane was incubated with caspase 3 primary antibody (1:1000 dilution; sc-7148 Santa Cruz Biotechnology Inc., Dallas, TX, USA) overnight at 4 °C, and then probed with horseradish peroxidase (HRP) conjugated secondary antibody (1:10,000 dilution; AbCam, Cambridge, UK) for 1 h at RT. Bound antibodies were detected with the use of Clarity Western ECL Substrate. The sample loading control was evaluated by a stain-free detection of proteins in the gel after electrophoresis.