Characteristics of replicative senescent HUVECs. (a) Senescent-associated β-galactosidase (SA-β-gal) activity staining of non-senescent and senescent HUVECs (bar 250 μm). (b) Immunofluorescent labeling of γH2AX with below the corresponding DAPI images to visualize the nuclei of all HUVECs (bar 25 μm). (c) Western blot analysis of Cyclin D1 and p21 expression levels in cell lysates of non-senescent and senescent HUVECs. Cyclin D1 and p21 are typical senescence markers. Tubulin was used as a loading control.