Research Paper Volume 7, Issue 12 pp 1143—1155

Figure 4. Inverse correlation between p27 levels and NSun2 levels in replicative senescence. (A) Left, the levels of proteins NSun2, p53, p16, p27, CDC25C, CDK1, and GAPDH in early-passage (Proliferating ‘Young’, ‘Y’, ~PDL 28), middle-passage (middle, ~PDL 40), and late-passage (Senescent, ‘S’, ~PDL 55) human diploid fibroblasts (2BS) were assessed by Western blotting. Right, the levels of proteins NSun2, p27, and GAPDH in Proliferating (Y) and Senescent (S) IDH4 cells were analyzed by Western blot analysis. (B) RNA was prepared from young (Y, ~PDL 28) and senescent (S, ~PDL 55) 2BS cells as well as young (Y) and senescent (S) IDH4 cells and RT-qPCR analysis was performed to assess the levels of p27 and CDK1 mRNAs. (C) RNA described in Fig. 4B was subjected to methylation-specific PCR analysis to assess the methylation of p27 and CDK1 mRNA. Data are representative from 3 independent experiments. (D) The density of the methylation-specific PCR (% of Ctrl) in Fig. 4C relative to that of the mRNA levels (% of Ctrl) shown in Fig. 4B is shown. Data represent the means ± SD from 3 independent experiments; significance was analyzed by Student's t test.