Fig. 6. Antioxidants inhibit TGFß1-mediated transdifferentiation in dermal and skin equivalents. (A) Fibroblasts seeded for 2 days in the dermal equivalent (DE) were untreated or treated with NAC, selenite or Trolox before stimulation with rTGFß1. After a further 48 hours, the collagen gel was dissolved with collagenase, the cells lysed and subjected to western blot analysis for SMA. Detection of -tubulin confirmed equal loading. The diameter (in cm) of the contracted or non-contracted collagen lattices was used as a measure of the contractile force of the cells. Three independent experiments were performed. Bar, 1 cm. (B) Histological structure of a skin equivalent (HE staining). cc-GAG, collagen-chitosan-glycosaminoglycan; d, dermis; e, epidermis; f, fibroblasts; k, normal human epidermal keratinocytes; sc, stratum corneum. Bar, 25 µm. (C) Skin equivalents were incubated for 3 days with 10 ng/ml rTGFß1 keratinocyte-SFM medium (without supplements) or in combination with 5 mM NAC. After dispase II treatment, the dermis was homogenized and 50 µl clear lysate or sample subjected to western blot analysis. -tubulin was used as a loading control. Quantitative data were standardized to -tubulin and densitometric values represent fold increase over the control, which was set at 1.0. The image is representative of two independent experiments.