Figure 7.

Collagen secretion and deposition by fibroblasts in vitro are increased by the CRT-binding sequence of TSP1. A: Sircol assay of human foreskin fibroblasts treated once daily for two days in media with 2 μmol/L ascorbic acid with 10 pmol/L TGF-β1, 10 μmol/L hep I peptide, 10 μmol/L modified hep I peptide, or media with 2 μmol/L ascorbic acid (no treatment). Results are expressed as the mean fold increase normalized to cells receiving no treatment ± SD; n = 3. Dashed lines in A–C indicate normalized value for no treatment. B: Sircol assay of human foreskin fibroblasts treated daily for two days with media with 2 μmol/L ascorbic acid and 10 nmol/L TSP1 or TSP1 incubated with either 20-fold molar excess of peptide CRT 19.36, or control peptide CRT 20.30A. Results are expressed as the mean fold increase normalized to cells receiving no treatment ± SD; n = 4. C: MEFs were treated daily for three days in media with 0.5% FBS, 2 μmol/L ascorbic acid with 30 nmol/L NoC1 with either 25-fold molar excess of peptide CRT 19.36, or control peptide CRT 20.30A. Soluble collagens were quantified by the Sircol assay. Results are expressed as the mean fold increase in soluble collagen normalized to cells receiving no treatment ± SD as analyzed by one-way analysis of variance; n = 4 separate experiments. D: MEFs were treated daily for four days in media with 2 μmol/L ascorbic acid with 10, 30, or 90 nmol/L NoC1 or with 20 μmol/L ascorbic acid. Cell layers were harvested with Laemmli buffer, separated by SDS-PAGE, and then immunoblotted with antibody to collagen III (top). Membranes were stripped and re-probed with anti-collagen I antibody (middle). Blots were stripped and reprobed with anti–β-tubulin as a loading control (bottom). *P < 0.05.