Figure 7.

Effect of CD44 gene silencing on TGF-β₁-dependent phenotypic activation. Confluent monolayers of young dermal fibroblasts were transfected with CD44 siRNA or scramble oligonucleotide control (scramble) for 48 hours. A: Total mRNA was extracted and CD44 mRNA expression was assessed by RT-Q-PCR. In parallel experiments after transfection the medium was 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 a further 72 hours, and α-SMA (B) or HAS2 (C) expression was assessed by RT-Q-PCR. Ribosomal RNA expression was used as an endogenous control, and gene expression was assessed relative to scramble (A) or control-scrambled (B and C) samples. The comparative C_T method was used for relative quantification of gene expression, and the results represent the mean ± SE of six individual experiments using cells isolated from two different donors. Statistical analysis was performed by Student's t-test: *P < 0.05; **P < 0.01. D–G: Immunohistochemical analysis was also performed to assess dermal fibroblast phenotype in untransfected cells (D and E) or cells transfected with either scramble oligonucleotide control (scramble) (F) or CD44 siRNA (G) before addition of serum-free medium with (E–G) or without (D) 10 ng/ml TGF-β1 for a further 72 hours. The cells were fixed and stained for α-SMA, mounted in Vectashield fluorescent mountant and viewed under UV light. Original magnification: ×100.