Gsk3787

THP-1, a human promonocytic cell line, was obtained from the American Type Culture Collection (ATCC; Manassas, VA). Cells were cultured in 10 cm tissue dishes in RPMI medium supplemented with 10% charcoal-treated fetal bovine serum, 2% nonessential amino acids, and 50 mg ml^-1 penicillin/streptomycin at 37°C in 5% CO₂. M. leprae was prepared from the footpads of nude mice as previously described [10 (link),11 (link)]. Live or heat-killed (80°C, 30 min) bacilli were added to cells, at typically multiplicity of infect (MOI) = 100 or otherwise indicated. Cells were further cultured for RNA or protein purification. GSK3787, BADGE, L-164,041, and dimethyl sulfoxide (DMSO) were purchased from Sigma Aldrich (Saint Louis, MO). GSK3787, BADGE and L-164,041 were dissolved in DMSO. Stocking solutions were diluted in culture medium by 10,000-fold to indicated working concentrations, and their treatment began 2 h prior to M. leprae infection.

The 4-Chloro-N-(2-]ethyl)benzamide (Gsk3787) (Sigma-Aldrich, Cat # G7423) was used to specifically inhibit the PPARδ function during the pre-implantation period of bovine embryonic development. A 5 mg powder of Gsk3787 was dissolved in DMSO to make 2.5 mM stock solution and was kept in the dark condition at −20 °C until use. Stock solution was serially diluted in phosphate buffered saline (PBS). COCs were randomly distributed into five groups (n = 50 to 55/group). Different concentrations of Gsk3787 (0 µM, 8 µm, 10 µM, 12 µM and 15 µM) were used in IVM and IVC medium. The study was performed in 10 to 11 reciprocal sets of independent experiments. Based on the blastocyst development and hatching rate, 15 µM dose showed maximum effect on PPARδ activity. For further analysis, 15 µM dose was used.