Figure 5.

Inhibition of APC/C promotes premature senescence in WI-38 fibroblasts.A, human diploid WI-38 fibroblasts were cultured for 10 days in the presence of Apcin (50 μM) and pro-TAME (40 μM). Treatment with DMSO served as control. Cells were collected and cell lysates were subjected to immunoblotting analysis using antibody probes specific for p16, p21, phospho-p53 (P-p53), γ-H2A.X, securin, and RAD21. Immunoblotting with anti-β-actin IgGs was performed as control. Ponceau S staining shows equal total protein loading. Quantification of protein band intensity is shown at the bottom of each blot. B–C, WI-38 cells were treated with Apcin (50 μM) and pro-TAME (40 μM) (A + T). After 10 days, cells were stained to detect senescence-associated β-galactosidase (SA β-gal) activity. Quantification is shown in (B), representative images are shown in (C). The scale bar represents 100 μm. D, WI-38 fibroblasts were treated with sublethal oxidative stress (450 μM H₂O₂ for 2 h) and recovered in complete medium in the presence of 1 μM AZ3146. WI-38 cells were recovered in the presence of DMSO as control. Cells were then subjected to senescence-associated β-galactosidase (SA β-gal) activity staining. Quantification of the percentage of cells that resulted positive to SA β-gal activity is shown. Values in (B) and (D) represent means ± SEM; statistical comparisons were made using the student’s t test. ∗p < 0.001. DMSO, dimethyl sulfoxide.