Figure 1.
Over-expression of Notch1 prolongs the lifespan of vascular endothelial cells.
(A) Western blot analysis of full length Notch1 (Notch1) and Notch intracellular domain (NICD) expression in human endothelial cells infected with a retroviral vector expressing Notch1 (N1OE) or the empty vector (Mock). GAPDH was used as the loading control. (B) Infected cells were passaged until senescence, and the total number of population doublings was determined (n = 3). (C) Senescence-associated β-galactosidase (SA-β gal) staining of endothelial cells prepared as in Figure 1A. Scale bar = 50 µm. The right graph shows quantitative data on SA-β gal-positive cells (n = 4). (D) Western blot analysis of p53, p21, and p16 expression in endothelial cells prepared as in Figure 1A. The right graph shows quantitative data on p53, p21, and p16 expression (n = 3). (E) Western blot analysis of full length Notch1 (Notch1) expression in human endothelial cells infected with a retroviral vector expressing Notch1 shRNA (N1KD) or sh-Control (Mock). (F) Infected cells were passaged until senescence, and the total number of population doublings was determined (n = 3). (G) Senescence-associated β-galactosidase (SA-β gal) staining of endothelial cells prepared as Figure 1E. Scale bar = 50 µm. The right graph shows quantitative data on SA-β gal positive cells (n = 4). (H) Western blot analysis of p53, p21, and p16 expression in endothelial cells prepared as in Figure 1E. The right graph shows quantitative data on p53, p21, and p16 expression (n = 3). All values represent the mean ± s.e.m. *P<0.05, **P<0.01.
Figure 2.
Over-expression of Jagged1 prolongs the lifespan of vascular endothelial cells.
(A) Real-time PCR analysis showing the relative expression of Notch ligands (DLL1, DLL3, DLL4, JAG1, and JAG2) in human endothelial cells (n = 5). (B) Western blot analysis of Jagged1 and Notch intracellular domain (NICD) expression in human endothelial cells infected with a retroviral vector encoding Jagged1 (J1OE) or an empty vector (Mock). Actin was used as the loading control. (C) Infected cells were passaged until senescence, and the total number of population doublings was determined (n = 3). (D) Senescence-associated β-galactosidase (SA-β gal) staining of endothelial cells prepared as in Figure 2B. The graph shows quantitative data on SA-β gal-positive cells (n = 4). (E) Western blot analysis of p53, p21, and p16 expression in endothelial cells prepared as in Figure 2B. The right graphs show quantitative data on p53, p21, and p16 expression (n = 3). (F) Western blot analysis of Jagged1 in human endothelial cells infected with a retroviral vector expressing human Jagged1 shRNA (J1KD) or shControl (Mock). (G) Infected cells were passaged until senescence, and the total number of population doublings was determined (n = 3). (H) Senescence-associated β-galactosidase (SA-β gal) staining of endothelial cells prepared as in Figure 2F. The graph shows quantitative data on SA-β gal-positive cells (n = 4). (I) Western blot analysis of p53, p21, and p16 in endothelial cells prepared as in Figure 2F. The right graphs show quantitative data on p53, p21, and p16 expression (n = 3). All values represent the mean ± s.e.m. *P<0.05, **P<0.01.
Figure 3.
Microarray analysis in human endothelial cells.
Microarray analysis of human endothelial cells showing that the Notch signaling positively regulates the expression of ID1 and DUSP1. OE; over-expression, KD; knock-down.
Figure 4.
Up-regulation of Id1 inhibits premature senescence induced by Notch1 disruption.
(A) Real-time PCR for the expression of ID1 in Notch1 over-expressing endothelial cells (N1OE), Notch1 knockdown cells (N1KD), and Mock-infected cells (Mock) (n = 12). (B) Western blot analysis of Id1 in endothelial cells prepared as in Figure 4A. The graphs display quantitative data on Id1 expression (n = 3). (C) Human endothelial cells were infected with a retroviral vector encoding Notch1 shRNA (N1KD) or an empty vector (Mock). Infected cells were then transduced with pLNCX-Id1 (Id1+) or an empty vector (Mock+) and subjected to the proliferation assay as described in the legend for Figure 1B (n = 3). (D) Western blot analysis of p16 and Id1 expression in endothelial cells prepared as in Figure 4C. The right graph displays quantitative data on p16 expression (n = 3). (E) Human endothelial cells were co-infected with retroviral vectors encoding Notch1 and Id1. Infected cells were passaged until senescence, and the total number of population doublings was determined (n = 3). (F) ChIP assay of the direct association between Notch1 and Id1 in Notch1 overexpressing cells (Notch1) or mock-infected cells (Mock) (n = 5). The amount of activated Notch1 localized to the CBF1-binding element was estimated by real-time PCR. The β-globin locus was used as a negative control. (G) Human endothelial cells were infected with a lentiviral vector encoding p16 shRNA (p16KD) or an empty vector (Mock). Infected cells were then transduced with a retroviral vector encoding Notch1 shRNA (N1KD) or an empty vector (Mock) and subjected to the proliferation assay as described in the legend for Figure 1B. (H) Senescence-associated β-galactosidase (SA-β gal) staining of endothelial cells prepared as in Figure 4G. The graph displays quantitative data on SA-β gal-positive cells (n = 4). All values represent the mean ± s.e.m. *P<0.05, **P<0.01.
Figure 5.
Inhibition of p38MAPK prevents induction of premature senescence by Notch1 disruption.
(A) Real-time PCR for the expression of DUSP1 (MKP1) in Notch1 over-expressing endothelial cells, Notch1 knockdown cells, and Mock-infected cells (n = 8). (B) Western blot analysis of phospho-p38MAPK (p-p38) and whole p38MAPK expression. The graphs show quantitative data on phospho-p38 expression (n = 3). (C) Human endothelial cells were infected with a retroviral vector encoding Notch1 shRNA (N1KD) or an empty vector (Mock). Infected cells were then treated with vehicle (Cont) or SB203580 (SB203580+) and subjected to the proliferation assay as described in the legend for Figure 1B (n = 3). (D) Western blot analysis of p16 and Id1 expression. The right graphs show the quantitative data on p16 and Id1 expression (n = 3). (E) Real-time PCR for the expression of ID1 (n = 6). (F) Human endothelial cells were infected with a retroviral vector encoding Notch1 shRNA (N1KD) or an empty vector (Mock) and were treated with vehicle (Cont) or proteasome inhibitor MG132 (MG132+), after which the expression of Id1 was assessed by western blot analysis. The right graph shows quantitative data on Id1 expression (n = 3). (G) Human endothelial cells were treated with vehicle (Cont) or anisomycin (Anisomycin+) for 0, 15 or 30 minutes, after which the expression of Id1, phosphorylated p38MAPK (p-p38), and whole p38MAPK was determined by western blot analysis. The right graph shows quantitative data on Id1 expression (n = 3). (H) Western blot analysis of the expression of Id1, phosphorylated p38MAPK (p-p38), and whole p38MAPK in human endothelial cells pre-incubated with vehicle (Cont) or MG132 for 1 hour followed by treatment with or without anisomycin for 15 minutes. (I) Flag-tagged Id1 over-expressing cells were pre-incubated with MG132 for 1 hour, followed by treatment with vehicle (Cont) or anisomycin (Anisomycin+) for 0, 15, or 30 minutes. Cell lysates were immunoprecipitated with FLAG M2 agarose. Then the levels of serine phosphorylated Id1 (p-Ser), threonine phosphorylated Id1 (p-Thr), and whole Id1 were assessed by western blot analysis (right; OUTPUT). Expression of Id1, phosphorylated p38MAPK (p-p38), and whole p38 MAPK was also estimated before immunoprecipitation (left; INPUT). All values represent the mean ± s.e.m. *P<0.05, **P<0.01.
Figure 6.
Up-regulation of Id1 improved the phenotypic changes induced by Notch1 disruption.
(A) Real-time PCR analysis showing the expression of Cdkn2a (p16) in the aortas of young mice (12–15 weeks) or old mice (50–55 weeks) (n = 6). (B) Real-time PCR analysis showing the expression of Notch1, ID1, and Cdkn2a (p16) in aortas prepared as in Figure 6C (n = 6). (C) Aortic ring assay performed in endothelial cell-specific Notch1 heterozygous knockout mice (N1KO) and their littermate controls (Cont) infected with lentivirus expressing human Id1 (lenti-Id1) or an empty vector (Mock). Cultured aortic rings were immunostained with BS1 lectin-FITC (Green). Scale bar = 100 µm. The graph displays the quantitative data for the number of sprouting cells (n = 14). All values represent the mean ± s.e.m. *P<0.05, **P<0.01.