Fig. 2.
pALTINK4a/b expression is induced by ECI, confluence, and senescence. (A) Naked mole rat fibroblasts were passaged under four different conditions. “Growing” cells were passaged at low cell density to ensure continuous cell proliferation. “HAase” cells were passaged at low cell density in the presence of hyaluronidase. “ECI” cells were kept in the same plate without passaging for 10–14 d until they stopped proliferation and entered the ECI state. “Confl.” cells were cultured in the presence of hyaluronidase until they reached confluence at high cell density. RNA was extracted from the cells, and expression from INK4a/b locus was analyzed by qPCR. (B) Naked mole rat cells were treated with indicated doses of UV-C and analyzed for expression of INK4a/b products as described above. (C) Naked mole rat cells were treated with 20 Gy of γ-irradiation, kept for 30 d until they developed senescent morphology, and analyzed for expression of INK4a/b products as described above. Four lines of low-passage naked mole rat skin fibroblasts were used for each experiment. Error bars indicate SD; *P < 0.05; **P < 0.005; Student’s t test.