Figure 4.

Inhibition of hyaluronidase (HYAL)-2 expression delays but does not prevent myofibroblast phenotype acquisition or transforming growth factor beta 1 (TGFB1; TGF-β1)–associated gene expression. Fibroblasts were grown to 50% confluence prior to transfection with negative control (scrambled) siRNA or with siRNA targeting HYAL2 expression (siHYAL2). Cells were growth-arrested for 48 hours before treatment with serum-free medium alone or serum-free medium containing 10 ng/mL TGF-β1 for 0, 24, 48, or 72 hours. A: Western blot was used to assess HYAL2 protein knockdown. B: Quantitative RT-PCR (RT-qPCR) was used to assess HYAL2 mRNA knockdown. C: Cells were then washed with phosphate-buffered saline, fixed, and permeabilized before staining to visualize α-smooth muscle actin (SMA). Images are representative of three individual experiments. D–F: RT-qPCR was also used to assess TGF-β1–regulated expression of ACTA2 (α-SMA) (D), extra domain-A–fibronectin (EDA-FN) (E), and TGFB1 (F) mRNA after incubation with either HYAL2 or scrambled siRNA. Data are expressed as means ± SEM of three independent experiments. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. Scale bars = 10 μm.