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. 2014 Jul 24;9(7):e102186. doi: 10.1371/journal.pone.0102186

Figure 2. CDC42 signaling regulates pro-inflammatory gene expression in senescent endothelial cells.

Figure 2

(A) 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 CDC42, siRNAs for PAK2, or control siRNA (siCont). Expression of pro-inflammatory genes was examined by real-time PCR after 72 hours. n = 3. (B) Human endothelial cells were infected with an empty vector (Mock) or a retroviral vector encoding p21 to induce senescence (p21) and were harvested at 6 days after infection. The pull-down assay for active CDC42 (GTP-CDC42) was performed as described in Methods. The graph indicates the relative level of active CDC42. n = 3. (C) Expression of phopho-PAK2 and total PAK2 was examined by western blotting in human endothelial cells prepared as in Figure 2B. (D) Human endothelial cells were infected with an empty vector (Mock) or a retroviral vector encoding active CDC42 (CDC42 V12), and expression of pro-inflammatory genes was examined by real-time PCR at 6 days after infection. n = 3. (E) Proliferation of endothelial cells infected with a retroviral vector encoding active CDC42 (CDC42 V12) or an empty vector (Mock). n = 3. (F) Human endothelial cells were infected with an empty vector (Mock) or a retroviral vector encoding active CDC42 (CDC42 V12). Six days after infection, the cells were transduced with 3 sets of siRNAs for RELA, siRNA for IKKs, or control siRNA (siCont). Expression of pro-inflammatory genes was examined by real-time PCR after 72 hours. n = 3. (G) Human endothelial cells were infected with a retroviral vector encoding p21 to induce senescence (p21-senescent). Six days after retroviral infection, the cells were infected with an adenoviral vector encoding a dominant-negative form of myc-tagged CDC42 (CDC42 N17) or LacZ. Expression of phospho-RELA (Ser536) and total RELA were examined by western blotting at 48 hours after adenoviral infection. (H) Expression of pro-inflammatory genes in endothelial cells prepared as in Figure 2G was examined by real-time PCR. n = 3. Data are shown as the mean ± SEM. *P<0.05, **P<0.01, ***P<0.001.