Fig. 3. Effects in HDFs at early cumulative population doublings (CPDs) to 10 exposures of UVB at 250 mJ/cm² per exposure on the steady-state mRNA level of senescence- and cell cycle-associated genes, and the level of intracellular and extracellular protein level of apo J. (A) Steady-state mRNA level of apolipoprotein J (apo J), fibronectin (fibro), osteonectin (osteo), SM22, p21^WAF-1, p53, c-fos and c-jun. Total RNA was extracted at 72 hours after the last stress. The GAPDH steady-state mRNA level was used as reference in the real-time RT-PCR. (a) The results obtained from the UVB-treated cells are expressed as percentage of the steady-state mRNA level of the respective mRNA species in control cells (CTL). (b) The results obtained with senescent cells (late CPDs) are compared with cells at early CPDs. Results are given as mean ± s.d. of three independent experiments. (B) Analysis of intracellular (a) and extracellular (b) protein level of apo J by western blotting. Proteins were extracted at 72 hours after the last stress. -tubulin protein was used as reference level to estimate the intracellular level of apo J while the same volume of collected medium was used to determine the extracellular levels. 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.