(A) Protocol for the induction of cellular senescence in salamander cells. A1 cells are induced to undergo cellular senescence upon UV irradiation combined with p53 stabilisation (1 µM nutlin3a). SA-β-gal positive cells (blue) appear at 7 days post-induction (dpi) and represent 80% of the total population by 12 dpi. (B) SA-β-gal staining of UV (10J/m2) irradiated cells at 12 dpi in the presence of the indicated compounds. (C–F) Quantification of SA-β-gal (C), BrdU (E), DHR123 (ROS) and γH2AX foci (F) positive cells, or gadd45 expression relative to ef1-α (D) at 7dpi following the indicated UV doses/treatments (n = 4). (G) SA-β-gal/EdU co-staining following subculture of proliferating or senescent cells in growth medium supplemented with 5 µM EdU. No SA-β-gal+/EdU+ cells are found, indicating cell cycle withdrawal of SA-β-gal+ cells (n = 5). Scale bar: 50 µM. (H) The cells identified by the Sa-β-gal staining exhibit other hallmarks of senescence such as persistent γH2AX foci (γH2AX immunostaining), high levels of ROS (DHR123), and extended mitochondrial (Mitotracker) and lysosomal (Lysotracker) networks. Lamin B1 is present at normal levels in senescent cells (representative images, n = 4). Scale bar: 50 µM. (I) Senescence-associated secretory phenotype analysis reveals similarities between salamander and mammalian senescent cells. Conditioned medium (SN) and whole cell extracts from A1 control (A1) or senescent (Sen) cells were analysed by cytokine/protease antibody arrays. The proteins whose levels vary significantly (p < 0.05) relative to A1 whole cell extracts are represented in the heat map. Asterisks indicate SASP factors upregulated in both salamander and mammalian senescent cells.