Figure 1

Effect of cCAF on TN levels in culture. (A-D) Embryonic connective tissue fibroblasts immunolabeled for TN. (A) In untreated cultures, some fibroblasts show a small amount of staining for this protein, which is characteristic of CEFs in culture. (B&C) Cultures treated for 3 days with cCAF (B) or with the N-terminal peptide (C) show that more fibroblasts stain for TN and that the staining is more intense than for untreated cells. (D) Treatment of cells with the C-peptide has a smaller effect on the number of cells staining for TN. (E) Immunoblot analysis for TN to quantify the results observed in (A-D). All lanes contained equal amounts of total protein, as measured by the DC protein assay (BioRad). Cells treated with cCAF or the N-peptide for 3 days show higher levels of TN than untreated or C-peptide treated cells. (F) Northern blot analysis of TN mRNA. Fibroblasts were treated with cCAF or its terminal peptides, total RNA extracted using TRIzol reagent and RT-PCR was performed as described in Materials and Methods. Amplification of TN mRNA reveals a substantial increase inTN mRNA with cCAF and N-peptide treatments whereas the C-peptide stimulated a small increase. To quantify the amount of RNA present, an internal control against 18S rRNA was used.