Clinical features and fundoscopy.

<p>Loose and sagging skin around the neck (A), legs (B), and back and hips (C). Peripapillary atrophy of the retina (arrow, D).</p

Histopathology of skin lesions.

<p>A biopsied specimen was stained with hematoxylin-eosin (A, B), Elastica van Gieson (C), von Kossa (D, G–I), and picrosirius red (E, F). The swollen collagen bundles were colored yellow−orange in polarizing microscopy (F). In the deeper dermis, the internal elastic lamina and a part of the arteriole walls were positive for calcification (G–I). The bar depicts 500 μm in (A–F) and 50 μm in (G–I).</p

Accelerated calcification and upregulation of ALP in GGCX dermal fibroblasts.

<p>(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. *: <i>P</i> < 0.05, **: <i>P</i> < 0.005, and ***: <i>P</i> < 0.0005. These experiments were performed at least twice, and a representative data set is shown.</p

Characterization of the mutated GGCX protein.

<p>(A) A homozygous deletion of T in the <i>GGCX</i> gene in the patient (Pt): c.2,221delT, p.S741Lfsx100. The wild-type DNA sequence is also shown (CON), and the peptide sequence is depicted at the top. (B) Western blotting. Long: long exposure; short: short exposure. (C) Immunofluorescence staining. The mutated and wild-type GGCX proteins (anti-GGCX) were identically localized at the Golgi apparatus (anti-58K Golgi). The bar depicts 50 μm (original magnification, ×40).</p

ALP expression and activity are higher in GGCX dermal fibroblasts than in PXE dermal fibroblasts.

<p>RT-PCR analysis of ALP mRNA levels (A) and analysis of ALP activities (B) without (open columns) or with (closed columns) osteogenic induction for 4 days in normal (CON, n = 3), PXE (PXE, n = 3), and GGCX (Pt) dermal fibroblasts. Values are mean ± SD for normal and PXE dermal fibroblasts and mean for GGCX dermal fibroblasts. The ALP mRNA expression levels in GGCX dermal fibroblasts were transferred from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177375#pone.0177375.g005" target="_blank">Fig 5F</a> for comparison. *: <i>P</i> < 0.05. These experiments were performed at least twice, and a representative data set is shown.</p

Expression of osteogenic trans-differentiation markers in GGCX dermal fibroblasts.

<p>RT-PCR analysis of BMP6 (A), RUNX2 (B), OSX (C), COL1A2 (D), POSTN (E), OCN (F), BMP2 (G), COL2A1 (H), and COL10A1 (I) in normal dermal fibroblasts (CON, n = 3) and GGCX dermal fibroblasts (Pt) at day 4 without (open columns) or with (closed columns) osteogenic induction. Values are mean ± SD for normal dermal fibroblasts and mean for GGCX dermal fibroblasts. *: <i>P</i> < 0.05. These experiments were performed at least twice, and a representative data set is shown.</p

Inhibition of BMP signaling blocks accelerated calcification.

<p>ALP staining of GGCX (Pt) dermal fibroblasts (A), ALP-positive cells (B), and ALP activity (C) of both normal (CON, n = 3) and GGCX (Pt) dermal fibroblasts treated with 2 μM dorsomorphin (DM(+)) for 4 days were reduced regardless of induction (ind (+) and ind (-)). The bar depicts 100 μm in (A). ALP-positive cells were counted in ten randomly selected fields. 1 field = 12.56 mm² (B). All assays were performed using three normal dermal fibroblast cell lines (CON1, CON2, and CON4) at day 4 without or with 2 μM dorsomorphin and without (open columns) or with (closed columns) osteogenic induction. Results are shown as mean ± SD. *: <i>P</i> < 0.05. These experiments were performed at least twice, and a representative data set is shown.</p