Figure 2

Effect of PPARγ and MAZ shRNAs applications on down-regulation of PPARγ1 expression and activity in the MCF-7 breast cancer cell line. A. To test the specificity of MAZ and PPARγ shRNAs for their target genes and estimate the efficiency of MAZ and PPARγ knock-down, Real-time PCR analysis of MCF-7 cells transfected with scrambled, MAZ, or PPARγ shRNA was performed. The fold change in gene expression was calculated using the ΔΔCt method. 18S was included as an internal control.B. Representative Western blot analysis of PPARγ1 expression in MCF-7 cells transiently transfected with scrambled, MAZ, or PPARγ shRNA. Densitometry was used to quantify PPARγ1 expression (n = 3). PPARγ1 expression is shown as a fold change in band intensity relative to control MCF-7 cells. Intensity of each band was normalized to actin. C. PPRE-mediated reporter activity was measured in MCF-7 cells transiently transfected with a 3XPPRE-mTK-pGL3 reporter plasmid and then co-transfected with MAZ or PPARγ shRNA expression plasmids. Cells were also subsequently treated with 10 μM Rosi for 20 hours. Data is expressed as mean fold change in luciferase to renilla ratios compared to control.