Synergistic Antiproliferative Effects of Combined γ-Tocotrienol and PPARγ Antagonist Treatment Are Mediated through PPARγ-Independent Mechanisms in Breast Cancer Cells
Figure 9
Western blot was performed to determine effect of treatment of γ-tocotrienol and rosiglitazone alone or in combination in PPARγ siRNA transfected (a) MCF-7 and (b) MDA-MB-231 cells. In addition, Western blot was performed to determine effect of treatment of γ-tocotrienol and GW9662 alone or in combination in PPARγ siRNA transfected (c) MCF-7 and (d) MDA-MB-231 cells. Cells were plated at a density of 05 cells/well (3 replicates per group) in 6-well plates in 2 mL antibiotic-free media. Transfections were performed using 5 μL lipofectamine 2000 according to the manufacturer’s protocol. Briefly, for each well to be transfected, 100 pmol of the scrambled or PPARγ siRNAs was diluted with 2 mL of media. After 6 h transfection, the medium was replaced with fresh growth media containing 10% FBS and cells were cultured for 18 h. Cells were then exposed to control or treatment media for a 4-day culture 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). * as compared with vehicle-treated controls.
