Fig 5. Accelerated calcification and upregulation of ALP in GGCX dermal fibroblasts.

(A) There were more mineral particles in GGCX dermal fibroblasts (Pt) than in normal dermal fibroblasts (CON) after 7 and 14 days of osteogenic induction. (B) Particles were positive for von Kossa staining at 11 days after induction. (C) ALP staining of normal and GGCX dermal fibroblasts at day 4 with (+) or without (-) induction. Periosteoblasts (POB) were positive controls for osteogenic induction. The bar depicts 100 μm in (A, C) (original magnification, ×40) and 20 μm in (B) (original magnification, ×100). (D) ALP-positive cells were counted in ten randomly selected fields. 1 field = 12.56 mm². (E) ALP activity analysis. Assays were performed using three different normal dermal fibroblast cell lines (CON1, CON2, and CON4) at day 4 without (open columns) or with (closed columns) osteogenic induction. Results are shown as mean ± SD. (F) Real-time PCR analysis of ALP. Assays were performed using three different normal dermal fibroblast cell lines (CON1–3) at day 4 without (open columns) or with (closed columns) osteogenic induction. Results are shown as mean ± SD. *: P < 0.05, **: P < 0.005, and ***: P < 0.0005. These experiments were performed at least twice, and a representative data set is shown.