Gw9662

Beta-catenin activator LiCl was obtained from Sigma-Aldrich (St. Louis, Missouri, USA) and used in 10 mM concentration [41 (link)]. Purified, recombinant human Wnt5a and Wnt11 (Chinese Hamster Ovary Cell Line, CHO-derived Gln38-Lys380) protein was purchased from R&D Systems (Minneapolis, USA) and used at a concentration of 1 μg/ml [37 (link)]. PPARgamma agonist rosiglitazone (RSG) and antagonist GW9662 were obtained from Sigma-Aldrich (St. Louis, USA) and used at 10 μM concentrations each [42 (link), 43 (link)]. During the experiments, A549 and F11 cells were treated with 10 mM LiCl (Sigma-Aldrich, St. Louis, Missouri, USA), 10 μM RSG and 10 μM GW9662 (Sigma-Aldrich, St. Louis, Missouri, USA) for 48 h.

To investigate the role of peroxisome proliferator‐activated receptor‐gamma (PPAR‐γ) pathway in ASD regulation of microglia phenotype, we used the PPAR‐γ inhibitor GW9662 (Sigma‐Aldrich). To investigate the potential role of the BDNF–tropomyosin receptor kinase B (TrkB) pathway in pro‐neurogenic effects of ASD, the K252a (Sigma‐Aldrich) was used to block the TrkB. GW9662 or K252a was dissolved in 0.9% saline containing 5% dimethyl sulfoxide (DMSO) at a concentration of 1 mg/mL and 2.5 μg/mL, respectively. After 3 weeks of CMS, five animal groups with similar sucrose preference and body weight were formed: control + saline + DMSO (Ctrl), CMS + saline + DMSO (CMS), CMS + ASD + DMSO (CMS + ASD), CMS + ASD + K252a (CMS + ASD + K252a), and CMS + ASD + GW9662 (CMS + ASD + GW9662). The mice were intraperitoneally administered saline, DMSO, ASD (40 mg/kg/day), and K252a (25 μg/kg/day) or GW9662 (1 mg/kg/day) once daily (at 16:00 h) for 3 weeks. The doses of K252a and GW9662 were chosen based on previous studies.⁹, ³⁵

M1 polarized macrophages were treated with either solvent or the PPAR-γ antagonist; GW9662 (10 µmol/L, Merck, Darmstadt, Germany), 2 h prior to repolarization to the M2c phenotype using TGF-β1. Thereafter, cells were incubated in RPMI medium supplement with 0.1% BSA (37 °C, 5% CO₂) and containing pHrodo Red Zymosan bioparticles (10 μg/mL, Invitrogen). After 30 min the cells were washed to remove nonphagocytosed material and zymosan uptake was visualized and quantified using an automated live cell imaging system (IncuCyte, Sartorius, Göttingen, Germany).

TGZ and pioglitazone were purchased from LKT Laboratories (St. Paul, MN) and BioVision (Milpitas, CA), respectively, and were dissolved in dimethyl sulfoxide (DMSO, Nacalai Tesque, Kyoto, Japan) just before use. The final DMSO concentration in the media did not exceed 0.1%. GW9662 (PPARγ inhibitor), SP600125 (JNK inhibitor), and SB202190 (p38 inhibitor) were obtained from Merck Millipore (Billerica, MA).

Murine fibroblast NIH3T3 cell lines were purchased from the American Type Culture Collection (ATCC; CRL-1658). The cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM, Gibco) supplemented with 10% bovine calf serum (BCS, Gibco) at 37°C and 5% CO₂. The cells were seeded in 6-well plates. The next day, fibroblasts were starved (without serum) for 12 h and then treated with different compounds. To elevate the role of EETs in fibroblast activatioin, we constructed an adenovirus-CYP2J2. Cells were infected with adenovirus-CYP2J2 (1 × 10⁹/mL) or vector (1 × 10⁹/mL) with the complete growth medium for 48 h and then incubated with or without TGF-β1 (5 or 10 ng/mL, Sino Biological, China). Fibroblasts were exposed to 5,6-EET, 8,9-EET, 11,12-EET, 14,15-EET (0.1, 0.5, 1 μM, Cayman, USA), or ethyl alcohol (1 : 300) for 5 min and then treated with TGF-β1 (5, 10 ng/mL) or vehicle control for 0.5 h to activate Smad2/3 signaling, for 24 h to active the p-PPARγ, or for 48 h to induce the proliferation and differentiation. The cells were exposed to GW9662 (a PPARγ inhibitor, 10 μM, Merck, USA) or dimethyl sulfoxide (DMSO, Sigma-Aldrich) for 1 h with the following treatment of 14,15-EET and TGF-β1.