Figure 3.

Overexpression of EGF-R in aged dermal fibroblasts. Aged dermal fibroblasts were transfected with either EGF-R-pCR3.1 (EGF-R transfected) or GFP (mock transfected). Transfection efficiency of EGF-R-pCR3.1 was 41.5%. Total mRNA was extracted and EGF-R (A) and α-SMA (B) mRNA expression was assessed by RT-Q-PCR. Ribosomal RNA expression was used as an endogenous control, and gene expression was assessed relative to mock transfected. The comparative C_T method was used for relative quantification of gene expression, and the results represent the mean ± SE of nine individual experiments using cells isolated from three different donors. Statistical analysis was performed by Student's t-test: **P < 0.01. N/S, not significant. C: EGF-induced proliferation was determined by incorporation of [³H]thymidine. Expression plasmids for EGF-R or GFP (mock transfected) were introduced into aged fibroblasts using Nucleofector technology before metabolic labeling, performed by incubation with 1 μCi/ml d-[³H]thymidine after addition of either serum-free medium alone (black bars) or serum-free medium containing 10 ng/ml EGF (gray bars) for 24 hours. Data are the mean ± SE from six individual experiments, using cells isolated from two different donors. Statistical analysis was performed by Student's t-test: *P < 0.05.