Measuring collagen deposition by gingival fibroblasts by traditional hydroxyproline analyses [Hong et al., 1999]. Bound dye was eluted and quantitated by spectrophotometry as described in Methods and Components. TGF-1 treated cultures served as optimistic controls. Information in Figure 1A show that 50 125 ng/ml CCN2/CTGF significantly Brd Inhibitor medchemexpress enhanced Sirius red dye binding (p 0.05), whereas ten and 25 ng/ml CCN2/CTGF had been unable to stimulate Sirius red dye binding to cell layers. TGF-1 strongly and substantially stimulated Sirius red binding. These data suggest that CCN2/CTGF stimulates collagen deposition at 50 ng/ml and greater, and that the effect of CCN2/CTGF is weaker than that of TGF-1. Staining in the same cell layers with all the DNA dye crystal violet followed by elution and spectrophotometric quantitation [Kostenuik et al., 1997] didn’t reveal consistent important increases induced by CCN2/CTGF indicating that cell quantity was not improved by CCN2/CTGF treatment (Table I). By contrast TGF-1 enhanced crystal violet binding to cell layers as expected, as TGF-1 is a potent mitogenic aspect for human fibroblasts cultured below these circumstances (Table I) [Clark et al., 1997]. Hence, CCN2/CTGF increases collagen deposition without the need of drastically stimulating growth of gingival fibroblast cultures. As a way to independently confirm that collagen deposition is improved by CCN2/CTGF, we cultured confluent cells as before within the constant presence of ten ng/ml TGF-1 or 100 ng/ml CCN2/CTGF, or no additions for seven days. Cell layers were collected as described inJ Cell Biochem. Author manuscript; offered in PMC 2006 May well 15.Heng et al.Page”Methods and Materials” and had been then hydrolyzed in 6 N HCl for 24 hours, and residues were analyzed for hydroxyproline levels. Results in Figure 1B show that TGF-1 and CCN2/CTGF increased hydroxyproline levels by 41.7 and 16.1 , respectively. Collagen deposition assays were reproducible involving experiments, and CCN2/CTGF generally enhanced Sirius Red staining of cell layers in all experiments, and more than 20 experiments have already been carried out. CCN2/CTGF stimulation of collagen deposition varied between 10 and 25 in different experiments, and collagen deposition was regularly stimulated by CCN2/CTGF. Changing serum lots affected the absolute worth of Sirius Red staining, but did not alter the finding that CCN2/CTGF stimulated collagen deposition. Data in Figure 1C carried out with the exact same cells as Figures 1A and B but having a various lot of newborn calf serum COX-1 Inhibitor drug showed that CCN2/CTGF nevertheless stimulated collagen deposition, and this impact was dosedependent. Studies in Figure 1A had been performed with gingival fibroblasts cultured from one particular individual. So as to determine that these experiments are representative of standard human gingival fibroblasts we measured CCN2/CTGF stimulated collagen deposition within a culture derived from a distinct donor. As seen in Figure 1D, CCN2/CTGF stimulated collagen deposition as determined by the Sirius red assay, and consistent with previous studies by our laboratory [Hong et al., 1999]. Structure/function studies The N-terminal half of CCN2/CTGF stimulates collagen synthesis, whereas the C-terminal half of CCN2/CTGF stimulated cell proliferation within a rat kidney cell line [Grotendorst and Duncan, 2005; Grotendorst et al., 2001]. Based on antibody inhibition research in vivo, the active portion of CCN2/CTGF in stimulating tooth improvement resides inside the N-terminal half of CCN2/CTGF [Shimo et al.,.
