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. 2012 Sep 18;7(9):e45331. doi: 10.1371/journal.pone.0045331

Figure 3. SIRT1 negatively regulates N1IC by deacytelation and regulates angiogenic activity in lung cancer-derived ECs.

Figure 3

(A) Lung cancer-derived ECs were transfected with control or SIRT1 siRNAs for 48 h and cultured in the absence or presence of DLL4 for an additional 6 h. Then, N1IC protein expression was detected using western blot analysis. siRNA-mediated blockade of SIRT1 activity in endothelial cells increased the endogenous N1IC protein levels. (B) Lung cancer-derived ECs were transfected with HA-N1IC, pcDNA-SIRT1, pcDNA-SIRT1 H363Y, or pcDNA3 (4 µg/µL each) for 48 h and were then treated with or without 5 nM NAM for an additional 12 h. Afterward, the cell extracts were immunoprecipitated with an anti-HA antibody and subjected to western blot analysis with an anti-acetyl lysine (upper panel) or anti-HA antibody (lower panel). (C) Lung cancer-derived ECs were triple transfected with N1IC, the acetyltransferase p300, and increasing amounts of SIRT1, and the cells were lysed 48 h later. Comparable levels of N1IC were immunoprecipitated and blotted for acetylated lysine residues (IP: HA and IB: anti-acetyl; upper panel). The blot was reprobed for HA, which demonstrated approximately equal levels of HA-N1IC (IP: HA and IB: HA; lower panel). (D) Lung cancer-derived ECs were transfected with N1IC, the acetyltransferase p300, and increasing amounts of SIRT1 together with the Renilla luciferase reporter and the pGL3-CBF plasmid containing a firefly luciferase reporter gene for 24 h. Then, luciferase activity was measured using a dual-luciferase reporter assay. The data were normalized to the Renilla luciferase activity (mean ± SD; n = 3). (E) qChIP analysis of SIRT1 occupancy at the Notch1 proximal promoter region in lung cancer-derived ECs. SIRT1 occupied a specific region at −500 bp of the Notch1 locus. SIRT1 was immunoprecipitated (IP) with anti-SIRT1 sera (SIRT1 IgG) or preimmune sera (normal IgG, used as a control) (mean ± SEM; n = 3). * P<0.05 as compared to the control. (F) Luciferase reporter gene assay results. pGL3, pGL3-Notch1 −500 to +400 (N+400), or pGL3-Notch1+400 to +1750 (N+1750) were transfected into HEK293 cells with or without SIRT1. *P<0.05. The error bars represent the SEM. (G) Expression of the Notch target genes HEY1 and HEY2 were assessed in control siRNA- and SIRT1 siRNA-transfected lung cancer-derived ECs, which were subsequently transfected with empty vector or N1IC for up to 48 h. SIRT1-deficient endothelial cells displayed markedly enhanced HEY1 and HEY2 activity in response to N1IC overexpression. *P<0.05 as compared to the control. (H) Lung cancer-derived ECs were transfected with control or SIRT1 siRNAs and treated with DMSO or 20 mM NAM for 24 h, followed by incubation with Matrigel for an additional 5 h. Next, tube formation was evaluated using an inverted phase microscope. The bar graphs represent the densitometry results. * P<0.05 as compared to the control.