Research Paper Volume 14, Issue 7 pp 2966—2988

Figure 5. Effect of knockdown of ERK1 or ERK2 on cellular senescence induced by IGFBP5 knockdown. (A) Levels of Igfbp5, Mapk3 (ERK1), and Mapk1 (ERK2) mRNA. Knockdown was confirmed by an RT-qPCR method. N=5 in each group. ***P<0.001 by paired Student’s t-test. (B) Representative immunoblots for phospho-Thr202/Tyr204-ERK1/2 (pERK1 and pERK2), ERK1, ERK2, and β-actin. P2 MEFs were transfected with control siRNA or Igfbp5 siRNA followed by additional transfection with either control, Mapk3 (ERK1), or Mapk1 (ERK2) siRNA. Cells were analyzed at 48 after transfection. (C) Quantitative data for pERK1, pERK2, total ERK1 and total ERK2 levels normalized to β-actin. N=4-7. (D) Representative images of SA-β-GAL staining in cells treated as in (B). A white dotted line in each field was added to visualize the representative outline of the cell. Red arrows indicate cells positive for SA-β-GAL staining. Scale bar, 100 μm. (E) Summary of the percentage of SA-β-GAL-positive cells. N=5 from three independent experiments. (F) Summary of cell surface area. N=5. (G) Analysis of cell number per field (1449 μm x1087 μm) in MEFs treated as in (D). N=8 in each group. (H) Levels of Cdkn2a (p16 and p19) and Cdkn1a (p21) mRNA. N=8 in each group. *P<0.05, **P<0.01, ***P<0.001 by one-way repeated measures ANOVA with a Student-Newman-Keuls test (C, E–H). Data are represented as mean +/- SEM. a.u.: arbitrary unit. NS: not significant.