siRNA-mediated reductions in PPARγ increased HPAEC ET-1, proliferation, and miR-27a levels.

<p>HPAEC were treated with scrambled (SCR) or siRNA (100 nM) to PPARγ for 72 hours. HPAEC mRNA or proteins were then isolated. In selected studies, HPAEC were subjected to MTT assays of proliferation. <b>A.</b> qRT-PCR was performed demonstrating decreases in PPARγ mRNA levels following treatment with siRNA. Each bar represents the mean ± SE PPARγ or ET-1 relative to 9S in the same sample expressed as fold-change vs. cells treated with scrambled siRNA (SCR). n = 4–9, *p<0.05 vs. SCR. <b>B.</b> HPAECs were treated with 100 nM SCR or PPARγ siRNA, and proteins were subjected to Western blotting for PPARγ, ET-1, or β-actin (ACTB). As illustrated by these representative Western blots, PPARγ depletion reduced HPAEC PPARγ protein levels, and increased levels of ET-1. <b>C.</b> HPAEC were treated with 100 nM SCR or siPPARγ and subjected to MTT proliferation assays. n = 6. *p<0.05 vs. SCR. <b>D.</b> HPAECs were treated with SCR or siPPARγ, and miRNA was isolated and subjected to qRT-PCR analysis along with RNU6B. Each bar represents the mean ± SE miR-27a relative to RNU6B expressed as fold changed vs. SCR. n = 4, *p<0.05 vs. SCR. <b>E.</b> miR-27a levels were upregulated in lungs from endothelial-targeted PPARγ knockout (ePPARγ KO) mice. miR-27a levels in lungs from endothelial-targeted PPARγ KO mice were determined with qRT-PCR. Each bar represents the mean ± SE level of miR-27a relative to RNU6B expressed as fold-change vs. littermate controls (LitCon), n = 3–6. *p<0.05 vs. LitCon.