Research Article

Synergistic Antiproliferative Effects of Combined γ-Tocotrienol and PPARγ Antagonist Treatment Are Mediated through PPARγ-Independent Mechanisms in Breast Cancer Cells

Figure 5

(a) Western blot and (b) qRT-PCR analysis to determine effect of γ-tocotrienol, rosiglitazone, and GW9662 given alone or in combination on levels of COX-2 and PGDS in +SA cells. For Western blot analysis, +SA cells were plated at 06 cells/100 mm culture dish and treated with control or treatment media for 4-day incubation period. Afterwards, whole cell lysates were prepared from each treatment group for subsequent separation by polyacrylamide gel electrophoresis (50 μg/lane) followed by Western blot analysis. Scanning densitometric analysis was performed on all the blots done in triplicate and the integrated optical density of each bond was normalized with corresponding β-actin, as shown in bar graphs below their respective Western blot images. Vertical bars in the graphs indicate the normalized integrated optical density of bands visualized in each lane ± SEM (arbitrary unit). For qRT-PCR analysis, +SA cells were plated at a density of 03 cells/well (3 replicates per group) in 6-well plates and treated with control or treatment media for a 4-day culture period. Total RNA was extracted and first-strand cDNA was generated from total RNA for each sample according to the manufacturer’s instructions. COX-2, PGDS, and GAPDH were measured using Taqman technology. Changes in mRNA levels of COX-2 and PGDS were normalized to mRNA level of GAPDH and represented as bar graph. Vertical bars indicate the normalized Ct value ± SEM (Arbitrary Unit) in each treatment group. * as compared with vehicle-treated controls.