The suppressive role of troglitazone on telomerase is independent from PPARγ. A) MDA-MB-231 cells were exposed to either 10 μM GW9662 or 100 μM BADGE for 24 hours. Cells then were treated with 20 μM troglitazone in the presence of GW9662 or BADGE for another 24 hours. At the end of the incubation time, the expression level of hTERT was determined by real time RT-PCR. Values are expressed as the percentage of vehicle-treated controls (DMSO) in the respective treatment condition. Results shown are as mean ± SD and are representative of three independent experiments. B) Utilizing shRNA interference, the expression of PPARγ was inhibited with four different shRNA oligos (71, 72, 73, and 74) in the MDA-MB-231 cell line. To assess the specificity of shRNA oligos against PPARγ , MDA-MB-231 cells were transfected with scrambled oligo as a control. The mRNA level of PPARγ was examined by real-time RT-PCR. WT, non-infected cells; SCR, scrambled oligo; 71-74 shRNA oligos. C) The TRAP assay was used to examine the activity of telomerase in MDA-MB-231 cells in the absence of PPARγ (Oligo 71, 72, 73, 74) compared to non-infected MDA-MB-231 cells (WT) and cells transfected with a scrambled oligo which acted as non-silencing control shRNA sequence (SCR). (500 and 250 ng of cell lysate, respectively. D) MDA-MB-231 cells carrying silenced PPARγ by shRNA were treated with 20 μM troglitazone or the equal volume of DMSO for 24 hours and telomerase activity was measured using the TRAP assay (500 and 250 ng of cell lysate, respectively). C and D) I.C., the internal PCR amplification control. CHAPS, lysis buffer only as a negative control. HeLa, 500 cell equivalent lysate as a positive control. Result shown is representative of two independent experiments.