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Fig. 2. Effect of a series of 10 exposures of HDFs at early cumulative population doublings (CPDs) to UVB at 250 mJ/cm2 on SA ß-gal activity, common 4,977 bp mitochondrial DNA deletion, incorporation of [3H]thymidine and the protein levels of p53, p21WAF-1 and p16INK-4A. (A) Proportion of cells positive for the SA ß-gal activity at 72 hours after 10 exposures to UVB at 250 mJ/cm2. Cells at early CPDs before exposure to UVB (d0), incubated 10 times in a thin layer of PBS without (CTL) or with UVB exposure (UVB) and cells at late CPDs (±90% of their proliferative life span) were examined. Results are given as mean ± s.d. of 3 independent experiments. (B) Detection of the common 4,977 bp mitochondrial DNA deletion by nested PCR after 10 exposures of UVB at 250 mJ/cm2. (a) Detection of a conserved 247 bp fragment of the mitochondrial DNA in cells incubated 10 times in a thin layer of PBS exposed to UVB (UVB) or not (CTL). Cells at day 0 (d0, before any incubation in PBS) were also examined. (b) Detection of the deletion by nested PCR. The 4,977 bp deletion is detected (404 bp product) in UVB-exposed cells (UVB) and not in CTL or d0 cells. (C) Effect of repeated UVB stresses on the incorporation of [3H]thymidine. Skin HDFs at early CPDs were incubated 10 times in a thin layer of PBS exposed to UVB (250 mJ/cm2) or not (CTL). They were trypsinised after 24, 48 or 72 hours after the last stress and incubated for 48 hours with [3H]thymidine at 1 µCi. Results are given as mean ± s.d. of three independent experiments. (D) (a) Analysis of p53, p21WAF-1 and p16INK-4 protein level by western blotting. Proteins were extracted at 72 hours after the last stress. 
-tubulin protein was used as reference level. (b) Quantification of the protein level. The results are expressed as 100% of the protein level in control cells (CTL). Results are given as mean ± s.d. of three independent experiments. Statistical analysis was carried out with the Student's t-test. ns, non-significant (P>0.05); *, 0.05>P>0.01; **, 0.01>P>0.001; ***, P<0.001.
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