NF-κB signaling regulates pro-inflammatory gene expression in senescent endothelial cells.

<p>(A–C) Human endothelial cells were infected with an empty vector (Mock) or a retroviral vector encoding cyclin-dependent kinase inhibitor 1A (p21) or cyclin-dependent kinase inhibitor 2A (p16) to induce senescence. Expression of pro-inflammatory genes, such as the chemokine (C–C motif) ligand 2 (<i>CCL2</i>), E-selectin (<i>SELE</i>), and vascular cell adhesion molecule 1 (<i>VCAM1</i>), in senescent endothelial cells was examined by real-time PCR at 6 days after infection with the retroviral vector for p16 (A) or p21 (B) or after mock infection. Expression of pro-inflammatory genes was also examined in human endothelial cells undergoing replicative senescence (C). We defined cells with replicative senescence as cultures that did not show an increase in cell numbers and remained subconfluent for 2 weeks. n = 5. (D) Human endothelial cells were infected with an empty vector (Mock) or a retroviral vector encoding p21 to induce senescence (p21-senescent). Six days after infection, the cells were transduced with 3 sets of siRNAs for <i>RELA</i> (1–3), siRNAs for IKKs (α, β, γ subunits), or control siRNA (siCont). Expression of pro-inflammatory genes was examined by real-time PCR after 72 hours. n = 3. (E) Expression of RELA in the nuclear and cytoplasmic fractions and phospho-RELA in whole cell lysates was examined by western blotting at 6 days after retroviral infection. Samples were prepared as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102186#pone-0102186-g001" target="_blank">Figure 1B</a>. Histone H3 expression served as the internal control for nuclear extracts and the level of nuclear RELA relative to Histone H3 was quantified. n = 3. Data are shown as the mean ± SEM. *P<0.05, **P<0.01, ***P<0.001.