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Fig. 7. TGF1-mediated signaling is altered in Sdc1-null keratinocytes. (A) Addition of increasing concentrations of TGF1 to wt and Sdc1-null keratinocytes inhibits keratinocyte proliferation with both genotypes showing identical responses. (B) A dual reporter assay was used to determine the fold induction of transcription of a TGF1-induced promoter (Smad4) compared with a control promoter (transketolase) 20 hours after wt and Sdc1-null keratinocytes were treated with 2 ng TGF1, as described in Materials and Methods. Note that the baseline of TGF1-mediated gene transcription for untreated keratinocytes was significantly higher for Sdc1-null keratinocytes, and that Sdc1-null keratinocytes had higher TGF1-induced gene expression at all concentrations of TGF1 tested; at 20 hours, these increases were significant for 250 and 500 ng TGF1. (C) Smad2 phosphorylation was measured directly in wt and Sdc1-null keratinocytes before and after TGF1 addition. In response to TGF1, wt keratinocytes showed an increase in P-Smad2, whereas Sdc1-null keratinocytes did not. (D) Amount of total TGF1 secreted into conditioned media was assessed via ELISA assay at the times indicated and data expressed as picogram (pg) per 10⁶ cells. (E) The amount of active TGF1 secreted into conditioned medium as well as in cell extracts was assessed using a standard mink lung epithelial reporter cell assay as described (see Materials and Methods); data are expressed in relative luciferase units.

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