Figure 3.

Downregulation of Cdc20 protein and mRNA expression occur in a p53-dependent manner.A and B, mouse embryonic fibroblasts (MEFs) derived from WT and p53 KO mice were treated with sublethal hydrogen peroxide (150 μM) for 2 h to induce premature senescence. Cells were washed with PBS and recovered in complete medium for 14 days. Untreated cells were used as control. Cells were then stained to detect senescence-associated β-galactosidase activity. Quantification is shown in (A), representative images are shown in (B). The scale bar represents 100 μm. Values in (A) represent means ± SEM; statistical comparisons were made using the student’s t test. ∗p < 0.001. C, p53 WT and p53 KO MEFs were subjected to oxidative stress as described in (A). After 14 days, Cdc20 mRNA levels were determined by RT-PCR using primers specific for Cdc20. RT-PCR using primers specific for LR32 was performed as internal control. Quantification of mRNA band intensity is shown at the bottom of the blot. D, p53 WT and p53 KO MEFs were subjected to oxidative stress as described in (A). After 14 days, cells were collected and cell lysates were subjected to immunoblotting analysis using antibody probes specific for Cdc20 and p53. Immunoblotting with anti-β-actin IgGs was done as control. Ponceau S staining shows equal total protein loading. Quantification of protein band intensity is shown at the bottom of the blot.