Analysis of expression and function of PPARα and PPARγ in Ph⁺ ALL cells. (A) Structure of compound TZD18. (B) RT-PCR analysis of the expression of PPARα. Total RNA was isolated, 0.2 μg each RNA sample was applied to RT-PCR, and specific amplification products for PPARα were visualized by electrophoresis and subsequent ethidium bromide staining. Total RNA extracted from the breast cancer cell line MDA-MB-231 was used as a positive control. (C) PPARα expression was determined by Western blot, as described in “Materials and methods.” Total protein isolated from the breast cancer cell line SKBR-3 served as a positive control for PPARα expression. (D) Functional analysis of several PPAR agonists and antagonists. SD1 cells were cotransfected with either a PPARα or a PPARγ expression vector (wt-PPARα or wt-PPARγ), a PPRE reporter vector (PPREx3-tk-luciferase), and a Renilla luciferase vector as an internal control for transfection efficiency. Eighteen hours after transfection, the cells were incubated with PPARα ligand (WY14,643), PPARγ ligand (PGZ), TZD18, PPARα antagonist (MK886), PPARγ antagonist (GW9662), or their combinations for another 24 hours. All drugs were used at 10 μM. After incubation, firefly and Renilla luciferase activities were determined. The firefly luciferase activity was normalized with Renilla luciferase activity and is shown as the fold increase over vehicle-treated controls. The result is a representative of 3 independent experiments.