Gsk3787

The procedure for collection and incubation of the porcine CL was previously described [66 (link)]. In the laboratory, the CL were dissected from the ovary, connective tissue was removed, and placed on ice in a sterile Petri dish. CLs were cut into small pieces (100 ± 10 mg, in duplicate from each experimental replicate). Each tissue explant was placed in M199 medium (Sigma Aldrich, St. Louis, MO, USA) supplemented with 0.1% BSA fraction V (Roth, Germany) and antibiotics. The explants were pre-incubated for 2 h in a water bath at 37 °C in an atmosphere of 95% O₂ and 5% CO₂. Then, the explants were treated with LPS (100 ng/mL, from E. coli) for 24 h. Explants not treated with LPS were considered as controls. The medium was removed, and the explants were incubated for 6 h with LPS alone or in combination with the PPAR β/δ ligands: GW0724 (agonist; 1 μmol/L or 10 μmol/L, Cayman Chemical Company, Ann Arbor, MI, USA) or GSK3787 (antagonist; 25 μmol/L, Cayman Chemical Company). Controls also contained dimethyl sulfoxide (DMSO, solvent for the tested PPAR ligands). After the incubation, tissue explants were frozen at −80 °C until further analysis.

C2C12 cells were obtained from Sigma (#91031101) along with Dulbecco's Modified Eagle Medium (DMEM; #D6429), fetal bovine serum (FBS; #F0926), horse serum (#H1270), and insulin (#I9278). Recombinant IL-15 was from GenScript (#Z03309-50) and GW-6471 (#11697) and GSK-3787 (#15219) were obtained from Cayman Chemicals. Trizol (#15596), SYBR green (#A25742) and a SuperScript VILO reverse transcription kit (#11754050) were purchased from ThermoFisher. The mitochondrial dye, Mito Red, was from Santa Cruz Biotechnology (SC-#301164). Male C57BL6 mice were kept on a 12 : 12 light dark cycle and fed a standard diet and water ad libitum. Gastrocnemius muscle was obtained following euthanasia. All animal procedures were approved by the Institutional Animal Care and Use Committee at Chapman University.