Figure 7.

Involvement of TSG-6 in TGF-β1-dependent fibroblast activation. A: Confluent monolayers of patient-matched young and aged dermal fibroblasts were growth-arrested in serum-free medium for 48 hours. The medium was then replaced with either serum-free medium alone (white bars) or serum-free medium containing 10 ng/ml TGF-β1 (black bars), and the incubations were continued for 72 hours. Total mRNA was extracted, and cDNA was prepared as described under Materials and Methods. TSG-6 expression was assessed by RT-QPCR. Ribosomal RNA expression was used as an endogenous control, and gene expression was assessed relative to control aged fibroblasts. The comparative C_T method was used for relative quantification of gene expression. Data are presented as the mean ± SE of six individual experiments with cells isolated from two patient donors. Statistical analysis was performed by Student’s t-test:*P < 0.05, compared with aged cells. N/S, not significant. B and C: To further examine the role of TSG-6, young dermal fibroblasts were transfected with TSG-6 siRNA or scrambled oligonucleotide control (scramble) and incubated in medium supplemented with 10% FBS for 24 hours. The medium was then replaced with serum-free medium for a further 24 hours, before addition of serum-free medium alone (white bars) or serum-free medium containing 10 ng/ml TGF-β1 (black bars) for 72 hours. Total mRNA was extracted and TSG-6 (B), and α-SMA (C) expression was assessed by RT-QPCR. Ribosomal RNA expression was used as an endogenous control, and gene expression was assessed relative to control scramble samples. 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, and statistical significance was taken as P < 0.05. N/S, not significant.