Cells (MCF7, MDA-MB-231, MDA-MB-468) are plated in 96-well plates at a density of 1×10³ cells per well in RPMI medium. After overnight incubation to allow for cell attachment, the medium is removed and replaced with fresh medium containing varying concentrations of BRL 49653 (1-100 μM), GW9662 (100 nM-50 μM) or solvent (DMSO) alone. MDA-MB-231 cells are also subjected to combinations of BRL 49653 (10, 50 μM) and GW9662 (1, 10 μM) added simultaneously. The final concentration of DMSO in all cases does not exceed 0.1% and is not found to be cytotoxic in any of the cell lines tested at this concentration. Chemosensitivity is assessed following a continuous 72 h exposure using a standard MTT assay.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Mice^[3]
Inbred C57BL/6 (B6, H2^b/b), DBA/1J (DBA/1, H2^q/q), FVB/NJ (FVB, H2^q/q) mice and congenic C.B10-H2^b/b/LilMcd (CB10, H2^b/b) mice are used. BADGE or GW9662, are administrated by daily intraperitoneal injection at 30 mg/kg for BADGE, or at 1 mg/kg for GW9662, from one day prior to the experiment and continued for up to 2 weeks. In the FVB AA model, some mice are injected with CsA (50 mg/kg/day) starting 1 hour after the LN injection, and continued for 5 days as immunosuppression. At the end of the experiments, the mice are euthanized by CO₂ inhalation.
Rats^[4]
Sixty-two male Wistar rats weighing 215 to 315 g are used in this study. Animals are randomly allocated into 6 groups as follows: (1) I/R group: control, rats are administered 10% (v/v) DMSO (vehicle for GW9662, 1 mL /kg, IP) 24 and 12 hours prior to renal I/R, and saline (vehicle for LPS, 1 mL /kg, IP) 24 hours prior to renal I/R (N=12); (2) I/R LPS group: rats are administered 10% (v/v) DMSO (vehicle for GW9662, 1 mL /kg, IP) 24 and 12 hours prior to renal I/R, and LPS (1 mg/kg, IP) 24 hours prior to renal I/R (N=11); (3) I/R GW9662 group: rats are administered GW9662 (1 mg/kg, IP) 24 and 12 hours prior to renal I/R, and saline (vehicle for LPS, 1 mL /kg, IP) 24 hours prior to renal I/R (N=9); (4) I/R LPS+GW9662 group: rats are administered GW9662 (1 mg/kg, IP) 24 and 12 hours prior to renal I/R, and LPS (1 mg/kg, IP) 24 hours prior to renal I/R (N=11); (5) Sham group: rats are subjected to the same surgical procedures as above, except for renal I/R. Rats are administered 10% (v/v) DMSO (vehicle for GW9662, 1 mL /kg, IP) and saline (vehicle for LPS, 1 mL /kg, IP) at times equivalent to those described above (N=12); (6) Sham GW9662 group: rats are subjected to the same surgical procedures as above, except for renal I/R. Rats are administered GW9662 (1 mg/kg, IP) and saline (vehicle for LPS, 1 mL /kg, IP) at times equivalent to those described above (N=7).

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Leesnitzer LM, et al. Functional consequences of cysteine modification in the ligand binding sites of peroxisome proliferator activated receptors by GW9662. Biochemistry. 2002 May 28;41(21):6640-50.  [Content Brief]

  [2]. Seargent JM, et al. GW9662, a potent antagonist of PPARgamma, inhibits growth of breast tumor cells and promotes the anticancer effects of the PPARgamma agonist BRL 49653, independently of PPARgamma activation. Br J Pharmacol. 2004 Dec;143(8):933-7.  [Content Brief]

  [3]. Sato K, et al. PPARγ antagonist attenuates mouse immune-mediated bone marrow failure by inhibition of T cell function.Haematologica. 2016 Jan;101(1):57-67.  [Content Brief]

  [4]. Collino M, et al. The selective PPARgamma antagonist GW9662 reverses the protection of LPS in a model of renal ischemia-reperfusion. Kidney Int. 2005 Aug;68(2):529-36.  [Content Brief]