Figure 1.

B‐myb was downregulated in the aortas of aged mice and human aortic endothelial cells (HAECs) undergoing replicative senescence. (A, B) The mRNA and/or protein expression levels of B‐myb, p53, p21, p16, pRb and cyclin D1 in the aortas of young (3 months) and aged (24 months) mice groups were analysed by real‐time PCR and western blotting. Each relative mRNA value was justified to the housekeeping gene GAPDH before normalization with that of the controls. Data are presented as mean ± SEM of three independent experiments. ∗ and ∗∗∗ indicate P<.05 and P<.001, respectively, between the two groups. A typical group of blots is shown and similar results were obtained in three separate experiments. GAPDH was used as a loading control. (C) Representative image of immunohistochemistry analysis performed with p16 antibody. A typical group of immunostaining is shown and similar results were obtained from young and aged groups. The percentage of senescent cells marked with p16 was calculated. Data are presented as mean ± SEM of three different samples. *** indicates P<.001 between the two groups. (D) Primary cultured HAECs at 15, 25, 30 and 48 population doubling (PDL) were stained by senescence‐associated β‐galactosidase (SA‐β‐gal). The SA‐β‐gal‐positive cells were directly observed under Nikon inverted microscope, and the representative images were shown (×100 magnification). After analysing SA‐β‐gal‐positive cells, the percentage rate is presented as mean ± SEM of three independent experiments. ** and *** indicate P<.01 and P<.001, respectively, compared with PDL15 cells. (E, F) The mRNA and/or protein expression levels of B‐myb, p53, p21, p16 and pRb in different PDLs cells were detected by real‐time PCR and western blotting. Each relative mRNA value was justified to the housekeeping gene GAPDH before normalization with that of the controls. Data are presented as mean ± SEM of three independent experiments. *, ** and *** indicate P<.05, P<.01and P<.001, respectively, compared with PDL15 cells. A typical group of blots is shown, and similar results were obtained in three separate experiments. GAPDH was used as a loading control