**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.