Research Article

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

Figure 8

Western blot analysis and luciferase assay to determine effect of γ-tocotrienol, rosiglitazone, and GW9662 given alone or in combination on PPARγ expression and activity in PPARγ siRNA transfected MCF-7 and MDA-MB-231 cells. For Western blot analysis, (a) MCF-7 and (b) MDA-MB-231 cells were plated at a density of 05 cells/well (3 replicates per group) in 6-well plates in 2 mL antibiotic-free media and allowed to adhere overnight. Transfections were performed using 5 μL lipofectamine 2000 according to the manufacturer’s protocol. 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 luciferase assay, (c) MCF-7 and (d) MDA-MB-231 cells were plated at a density of 04 cells/well (3 replicates per group) in 96-well plates and allowed to adhere overnight. After this cells were transfected with 32 ng of PPRE X3-TK-luc and 3.2 ng of Renilla luciferase plasmid per well and then cotransfected with scrambled or PPARγ siRNAs using 0.8 μL of lipofectamine 2000 transfection reagent for each well. After 6 h transfection, the media were removed; the cells were washed once and exposed to 100 μL of control or treatment media. Afterwards, cells were lysed with 75 μL of passive lysis buffer and treated according to manufacturer’s instructions using dual-glo luciferase assay system. Results were calculated as raw luciferase units divided by raw Renilla units. Vertical bars indicate PPRE mediated reporter activity ± SEM (arbitrary unit) in each treatment group. * as compared with vehicle-treated controls.