Fig 2: 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.

Fig 3: Cdc20 mRNA is downregulated in stress-induced and replicative senescent WI-38 fibroblasts.A, WI-38 fibroblasts were treated with sublethal hydrogen peroxide (450 μM) for 2 h to induce premature senescence. Cells were washed with PBS and recovered in complete medium for 14 days. Cells were collected and the expression level of Cdc20 mRNA was determined by RT-PCR using primers specific for Cdc20. Untreated cells were used as control. Expression of GAPDH was detected as internal control. B, human WI-38 fibroblasts were treated with sublethal UV-C light (10 J/m2) to induce premature senescence. Cells were then recovered in complete medium for 14 days. Cells were collected and the expression level of Cdc20 mRNA was determined by RT-PCR using primers specific for Cdc20. Untreated cells were used as control. Expression of GAPDH was detected as internal control. C, WI-38 cells were treated with 2% cigarette smoke extracts (CSEs) for 14 days to induce premature senescence. Cells were collected and the expression level of Cdc20 mRNA was determined by RT-PCR using primers specific for Cdc20. Untreated cells were used as control. Expression of GAPDH was detected as internal control. D, lysates from nonsenescent (Ctl) and replicative senescent human WI-38 cells were subjected to immunoblotting analysis using anti-Cdc20 IgGs. Lysate from oxidative stress–induced premature senescent WI-38 cells was used as positive control. Anti-β-actin IgGs were used to show equal total protein loading. Quantification of protein band intensity is shown at the bottom of the blot. E, expression of Cdc20 mRNA in nonsenescent (Ctl) and replicative senescent WI-38 fibroblasts was determined by RT-PCR using primers specific for Cdc20. Cdc20 mRNA expression in oxidative stress–induced senescent WI-38 cells served as positive control. Expression of GAPDH was detected as internal control. Quantification of mRNA band intensity is shown at the bottom of each blot.

Fig 4: Expression of CDC20 is associated with cSCC progression. (A) Normal skin (n=32) (magnification, ×100). (B) Actinic keratosis (n=31) (magnification, ×100). (C) cSCC in situ (Bowen's disease; n=27) (magnification, ×100). (D) Well-differentiated cSCC (n=29) (magnification, ×100). (E) Moderately or poorly differentiated cSCC (n=25) (magnification, ×100). (F) Semiquantitative analysis of CDC20 expression in the different tissues. CDC20, cell division cycle 20; cSCC, cutaneous squamous cell carcinoma; wd, well-differentiated; md/pd, moderately or poorly differentiated. *P<0.05, ***P<0.001 vs. normal skin.

Fig 5: Cdc20 and securin protein expression is downregulated in oxidative stress–induced senescent WI-38 fibroblasts.A, human diploid WI-38 fibroblasts were treated with sublethal hydrogen peroxide (450 μM) for 2 h. Cells were washed with PBS and recovered in complete medium for 14 days. Cells were collected and cell lysates were subjected to immunoblot analysis using protein-specific antibody probes. Untreated cells were used as control. Ponceau S staining shows equal total protein loading. Quantification of protein band intensity is shown at the bottom of each blot. B–D, premature senescence was induced in WI-38 cells as described in (A). Cells were then stained to detect senescence-associated β-galactosidase activity. Representative images are shown in (B), quantification is shown in (C). The scale bar represents 100 μm. The percentage of cells possessing enlarged and flat morphology (senescence-associated (SA) cell morphology) is shown in (D). Values in (C) and (D) represent means ± SEM; statistical comparisons were made using the student’s t test. ∗p < 0.001.