Figure 4.
Incorporation into the ECM and biosynthesis of collagen by NB1RGB cells treated with CNP and TGFβ. A, immunofluorescent staining for type I collagen. Cells were cultured for 72 h in the presence of CNP and TGFβ as indicated, and then immunostaining was performed. B, quantification of collagen by measurement of the pixels of the immunostaining pictures (mean ± S.D. of 10 fields; **, p < 0.05). C, immunofluorescence staining for type III collagen. Cells were cultured and treated as above, and then immunostaining was performed. D, quantification of collagen by measurement of the pixels of the immunostaining pictures (mean ± S.D. of 10 fields; **, p < 0.05). E, negative control of immunostaining, where nonimmune rabbit IgG was used in place of primary antibody. The picture was taken with a longer exposure. F, Western blotting for type I collagen. Cells were treated with CNP and TGFβ as above, and cells/ECM were collected and applied to Western blotting. The bands of α1 and α2 chains of type I collagen are indicated by arrows. The numbers below indicate the level of band density, with a nontreated sample as 100. G, quantification of collagen by SilcolTM assay (n = 3, mean ± S.D.; **, p < 0.05). H, biosynthesis of total proteins and collagens. Cells were treated with CNP and/or TGFβ and then labeled with [3H]proline for 24 h. Radioactivity incorporated in the cells was measured. Total protein levels (left panel, n = 3, mean ± S.D.; **, p < 0.05) and collagen synthesis levels (percent) of total proteins (right panel) are shown. The experiments were performed twice (G), three times (C, D, F, and H), or five times (A, B, and E) with essentially the same results. Representative results are shown.