Gw0742

PPARγ DNA binding activity was measured using a PPARγ transcription factor assay kit (Cayman Chemical, USA). Briefly, 10 μg of extracted nuclear proteins were added to wells containing immobilized dsDNA sequences corresponding to the peroxisome proliferator response element. Bound PPARγ was detected by the addition of specific antibodies against PPARγ. Relative PPARγ DNA binding activity was determined by normalizing the measurements obtained for cells transfected with Yulink-shRNA to those obtained for cells treated with Ctrl vector. Rosiglitazone (Santa Cruz), pioglitazone (Sigma), GW7647 (Sigma), and GW0742 (Sigma) were stored in the dark at − 20 °C. Working solutions were prepared by diluting the stock solution in media. Stable cells transfected with Yulink-shRNA or Ctrl vector were treated with or without agonist for 6 h, 12 h, or 2 days. RNA was subsequently isolated from cells using Quick-RNA MiniPrep (Zymo Research, USA), according to the manufacturer’s instructions.

GW0742 and GSK0660 were purchased from Sigma-Aldrich (St. Lois, MO, USA). Rosiglitazone, GW9662 and Bisphenol A diglycidyl ether (BADGE) are from Cayman Chemical Company (Ann Arbor, MI, USA). 5-bromo-2′deoxyuridine is from Sigma-Aldrich (St. Lois, MO, USA). Anti-PPARβ/δ and anti-Myc were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-PPARγ, anti-GFAP (Glial fibrillary acidic protein), anti-DCX (Doublecortin) and anti-SOX2 are from Cell Signaling Technology (Beverly, MA, USA). Anti-Nestin and anti-EGFR are from Millipore (Billerica, MA, USA), anti-Galactocerebroside C (GalC) was purchased from Sigma-Aldrich (St. Lois, MO, USA), anti-5-bromo-2′deoxyuridine is from Abcam (Cambridge, MA, USA) and anti-β III-Tubulin is from Promega (Madison, WI, USA). Restriction enzymes are all from New England Biolabs (Ipswich, MA, USA). GoTaq Flexi DNA Polymerase and RT-PCR reagents were purchased from Promega (Madison, WI, USA) and Invitrogen (Grand Island, NY, USA). siRNA-PPARβ/δ was purchased from Santa Cruz Biotechonology, siRNA-control and siGlo-Green Transfection Indicator were obtained from Thermo Fisher Scientific, Dharmacon Inc (Lafayette, CO, USA).

Dulbecco's modified Eagle's medium (DMEM) containing low or high glucose levels, fetal bovine serum (FBS), fetal calf serum (FCS), penicillin, and streptomycin were obtained from GIBCO (Grand Island, NY). Antibodies against C/EBPα, PPARγ, and β-actin were from Santa Cruz Biotechnology (Santa Cruz, CA). The RNA extraction kit was from Intron Biotechnology (Seoul, Korea). PPARγ, aP2, CD36, FAS, LPL, and GAPDH oligonucleotide primers were from Bioneer Co. (Daejeon, Korea). Rosiglitazone, pioglitazone, GW0742, GW7647, protein inhibitor cocktail, phenylmethyl sulfonylfluoride, hematoxylin,eosin and all other chemicals were from Sigma (St. Louis, MO). LFE and FSB were isolated from Lysimachia foenum-graecum as previously described [3 (link), 4 (link)].

The PPARβ/δ ligands GW0742, GSK3787, L−165042 and GSK0660 as well as 1400W, LPS O55:B5, sulfanilamide and naphthylethylenediamine dihydrochloride were purchased from Sigma (Gillingham, Dorset, UK). Sodium nitrate, orthophosphoric acid and DMSO were purchased from Fisher Scientific (Loughborough, UK). Primers from Applied Biosystem (Foster City, CA, USA): β-actin (Rn00667869_m1), Pdk−4 (Rn00585577_m1), Angptl−4 (Rn015228817_m1), Nos2 (Rn00561646_m1).

All the reagents, including insulin-transferrin-sodium selenite media supplement (ITS), recombinant human IL-1β, GW0742, nicotinamide, and EX527, were purchased from Sigma Chemical Company. Adenoviruses expressing PPARβ/δ (Ad-PPARβ/δ) and TTA (Ad-TTA) were kindly provided by Professor Nanping Wang of the East China Normal University Health Science Center, Shanghai, China. Adenoviruses expressing SIRT1 (Ad-SIRT1), SIRT1 siRNA (Ad-siSIRT1), and the control adenoviruse (Ad-siControl) were generously provided as gifts by Professor Yongsheng Chang of the Beijing Union Medical College, Beijing, China. Antibodies, including β-actin (Santa Cruz sc-47778, Dallas, TX, USA), COX-1 (Cayman Chemical 160109, Ann Arbor, MI, USA), COX-2 (Cayman Chemical 160106), PPARα (Santa Cruz sc-1982), PPARγ (Santa Cruz sc-1981), PPARβ/δ (Abcam ab8937, Cambridge, UK), and SIRT1 (Abcam ab32441) were used in the present study.