Research Paper Volume 14, Issue 7 pp 2966—2988

Figure 1. Downregulation of IGFBP5 in senescent MEFs. (A) Cumulative population doubling of MEFs during the 3T3 passage. N=8. (B) Representative images of senescence-associated β-galactosidase (SA-β-GAL) staining in MEFs at the 2nd (P2), 4th (P4), 6th (P6), and 8th (P8) passages. A white dotted line in each field was added to visualize the representative outline of the cell at each passage. Red arrows indicate cells positive for SA-β-GAL staining. Scale bar, 100 μm. (C) Summary data of the percentage of SA-β-GAL-positive cells. N=3 in each group. ***P<0.001 by one-way repeated measures ANOVA with a Student-Newman-Keuls test for multiple comparisons. (D) Summary data of cell surface areas. N=3. ***P<0.001 by one-way repeated measures ANOVA with a Student-Newman-Keuls test for multiple comparisons. (E) Levels of Cdkn2a (p16 and p19) and Cdkn1a (p21) mRNA in MEFs at P2 and P8. N=4 in each group. *P<0.05 by paired Student’s t-test. (F) Gene expression of IGFBPs in P2 and P8 MEFs. ND: not detected. N=5 in each group. *P<0.05, ***P<0.001 by paired Student’s t-test. (G) Representative immunoblots for p16, p19 and IGFBP5 in P2 and P8 MEFs. kDa: kilodalton. (H) Changes in Igfbp5 expression of senescent MEFs compared with young MEFs in five datasets from the Gene Expression Omnibus. (I) Changes in Igfbp5 and Cdkn2a (p16 and p19) mRNA levels during serial passage. N=4 in each passage. (J) Representative immunoblots (left) and quantitative data (right) for p21 and β-actin in P2, P4, P6, and P8 MEFs. N=3 in each passage from three independent experiments. *P<0.05, **P<0.01 by one-way repeated measures ANOVA with a Student-Newman-Keuls test. a.u.: arbitrary unit. Data are represented as mean +/- SEM. NS: not significant.