Research Paper Volume 14, Issue 7 pp 2966—2988

Figure 3. Effects of exogenous IGFBP5 on replicative senescence in MEFs. (A) Cumulative population doubling in MEFs passaged with a 3T3 method that were treated with a vehicle or IGFBP5 (10, 30 ng/ml) starting at P2 or 30 ng/ml IGFBP5 starting P4. N=5 in each treatment. *P<0.05, **P<0.01, ***P<0.001 by two-way repeated measures ANOVA with a Student-Newman-Keuls test. (B) Representative images of SA-β-GAL staining in MEFs at the 5th passage treated with the vehicle or IGFBP5 (30 ng/ml) starting at P2. 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=12 from three independent experiments in each treatment. *P<0.05 by unpaired Student’s t-test. (D) Summary data of the percentage of SA-β-GAL-positive cells in P4 MEFs transfected with an empty vector or IGFBP5-FLAG. N=8 from two independent experiments in each treatment. *P<0.05 by unpaired Student’s t-test. (E) Levels of Cdkn2a (p16 and p19) and Cdkn1a (p21) mRNA in P2 MEFs transfected with an empty vector or IGFBP5-FLAG. N=6 in each treatment. *P<0.05, ***P<0.001 by unpaired Student’s t-test. (F) Summary data of the percentage of SA-β-GAL-positive cells in P2 MEFs transfected with an empty vector or IGFBP5-FLAG. N=7-8 from two independent experiments in each treatment. *P<0.05 by unpaired Student’s t-test. Data are represented as mean +/- SEM.