The amino acid sequence of rat chondromodulin-I (rChM-I).

<p>Light gray circles indicate amino acid residues that are not conserved in human ChM-I (hChM-I). Circles in parentheses indicate four amino acid residues that are not conserved in mouse (mChM-I). Four thick bars connecting a pair of Cys residues indicate the intramolecular disulfide bonds, whose arrangements are assumed to be identical with those determined for bChM-I purified from fetal bovine cartilage <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094239#pone.0094239-Neame1" target="_blank">[27]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094239#pone.0094239-Hiraki3" target="_blank">[28]</a>. The putative glycosylation sites are also indicated. The arrowhead indicates the determined cleavage site that gives rise to the 14-kDa form of ChM-I.</p

Effects of 14-kDa ΔN-rhChM-I on the VEGF-A-induced chemotactic migration of HUVECs.

<p>Biological activity of G-rhChM-I and ΔN-rhChM-I was assessed by a modified Boyden chamber assay <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094239#pone.0094239-Miura1" target="_blank">[8]</a>. Serum-starved HUVECs (7×10⁴ cells) were preincubated for 30 min with G-rhChM-I (•) or ΔN-rhChM-I (▴), and seeded onto vitronectin-coated cell culture inserts in serum-free medium. Chemotactic migration of HUVECs was induced by the addition of VEGF-A (20 ng/ml) in the lower chamber, and the cells were allowed to migrate for 4 h toward VEGF-A. The number of cells that had migrated to the bottom surface of the insert was counted. Values are the percentages of migrated cell numbers in the control culture that was stimulated by VEGF-A, and are the means ± SD of a triplicate assay. The data are representative of three independent experiments with similar results.</p

Localization of MMP9 and MMP13 in mouse developing bone at E16.5.

<p>Frozen sections were prepared from legs of an E16.5 mouse embryo. Serial sections of proximal tibia were stained with MMP9 antibody (red; A), MMP13 antibody (red; B), hCHM-05 ChM-I MoAb (green; C) or N-ChM-I Ab (green; D). The nuclei were counterstained with DAPI (blue). Asterisks indicate the hypertrophic cartilage zone. Arrowheads indicate the boundary between cartilage and the invading front of vasculature. Scale bars, 100 μm.</p

Identification and immunoprecipitation of rat 14-kDa ChM-I.

<p>(A) 8 M urea extracts were prepared from male rat ribs (4-week-old) and subjected to immunoblotting with hCHM-05 ChM-I MoAb. Rat 20–25 kDa glycosylated ChM-I (asterisk) was detected as a broad band as well as a 14-kDa band (thick arrow) and a faint 17-kDa band (arrow). (B) Rat ChM-I extracted in 8 M urea buffer was immunoprecipitated with hCHM-05. Immunoprecipitates were resolved by SDS-PAGE and detected by silver staining. For the specification of immunoprecipitated bands (Ex + Ab), cartilage extracts without the antibody (Ex) or 8 M urea buffer with the antibody (Ab) were similarly processed during the immmunoprecipitation. The asterisk and the arrow indicate 20–25 kDa and 14-kDa ChM-I, respectively. (C) Primary cultures of rat costal chondrocytes were cultured to confluence for 3 days and then conditioned in DMEM/F12 medium in the presence of 10% FBS for the indicated periods of time. The collected conditioned medium was concentrated and subjected to immunoblotting with hCHM-05 ChM-I MoAb.</p

Differential distribution of 14-kDa ΔN-ChM-I and 20–25 kDa ChM-I in mouse developing bone at E16.5.

<p>Frozen sections were prepared from legs of an E16.5 mouse embryo. (A) Toluidine blue (TB) staining of the sagittal section of tibia and fibula. (B, C) Immunofluorescent views of the boxed area in panel A. Double immunofluorescent staining of the sagittal section of proximal tibia with CD31 (red) and N-ChM-I Ab (green; B) or hCHM-05 ChM-I MoAb (green; C). (D) Double immunofluorescent staining of the sagittal section of proximal tibia with N-ChM-I Ab (green) and hCHM-05 ChM-I MoAb (red). (E, F) Absorption test for hCHM-05 ChM-I MoAb is shown. The semiserial sections of tibia was immunostained with hCHM-05 ChM-I MoAb (green) preincubated with BSA (E) or G-rhChM-I (F). In panels D, E, and F, the nuclei were counterstained with DAPI (blue). Asterisks in the panels B-D indicate hypertrophic cartilage zone. Scale bars, 100 μm.</p

Identification by immunoblotting of a 14-kDa fragment of ChM-I in mouse cartilage extracts.

<p>(A) Cartilage extracts of 14-week-old male mice were prepared from ribs of wild-type (wt) and <i>Chm1</i> (-/-) null mice, and analyzed by immunoblotting with N-ChM-I Ab or hCHM-05 ChM-I MoAb. N-ChM-I Ab recognizes mouse 22-kDa glycosylated mature ChM-I (asterisks) as a broad immunoreactive band, whereas hCHM-05 ChM-I MoAb indicated the presence of a 14-kDa band (thick arrow) as well as a faint 17-kDa band (arrow). None of these bands were detected in the extracts from cartilage of <i>Chm1</i> (-/-) null mice. (B) Mouse tissue extracts were prepared from E11.5 whole embryos, and limbs dissected out at E13.5 and E16.5 as well as rib cartilage from 14-week-old male mice, and analyzed by immunoblotting with hCHM-05 ChM-I MoAb or β-actin antibody as a loading control. Note that the loading amount of rib cartilage extracts was reduced to 2.5 μg protein to give a quantitative range of signals in the same blot, while 10 μg protein was loaded per each lane for embryonic tissue extracts.</p

Illustration of Tnmd structure and design of antibody recognition position.

<p>Schematic illustration of the Tnmd structure. Domain structures are highlighted. The position of the antibody recognition site is indicated in red. The N-glycan glycosylation site is indicated by a hexangular box with the amino acid number. Conserved cysteine residues are indicated in capital letters. Transmembrane: transmembrane region; BRICHOS: BRICHOS domain; CS: possible cleavage site; CTD: C-terminal domain.</p

Determination of the subcellular localization of Tnmd.

<p>(<b>A</b>) Detection of Tnmd in cell fractions by western blotting. (<b>B</b>) Examination of the glycosylation of Tnmd with tunicamycin treatment. (<b>C</b>) Examination of the N-glycosylation of Tnmd with PNGase treatment. WL, whole cell lysate; Cytl: cytosol fraction; Mem: membrane fraction; Nuc: nuclear fraction; Cytn: cytoskeleton fraction. (<b>D-G</b>) The subcellular localization of the Tnmd protein in NIH3T3 cells transfected with Tnmd was examined by ICC. NIH3T3 cells were stained with cell organelle markers to indicate the plasma membrane [upper panels]. NIH3T3 cells transfected with Tnmd were double stained with cell organelle markers and the anti-Tnmd antibody [lower panels]. (<b>D</b>) Tnmd and plasma membrane marker, Pan-Cadherin (Cad). (<b>E</b>) Tnmd and Golgi apparatus marker, Wheat germ agglutinin (WGA). (<b>F</b>) Tnmd and β-Actin (Act). (<b>G</b>) Tnmd and α-Tubulin (Tub). SYTOX nuclear staining is shown in blue, cell organelle markers in green, and anti-Tnmd antibody in red. White arrowhead indicates the positive staining or organelle markers or anti-Tnmd antibody. Yellow arrowhead indicates the colocalization of the organelle marker signal and Tnmd signal. Scale bar = 50 µm.</p

Effect of Tnmd transfection on cell adhesion of PDL-h<i>TERT</i> cells.

<p>(<b>A</b>) Expression of Tnmd in PDL-h<i>TERT</i> cells transfected with a bicistronic vector carrying FLAG-tagged <i>Tnmd</i> or LacZ and Venus [pEBMulti-FLAG-Tnmd-2a-Venus, pEBMulti-LacZ-2a-Venus], as determined by western blotting. FLAG-tagged Tnmd was detected by anti-FLAG antibody, and Venus was detected by anti-GFP antibody. (<b>B</b>) Effect of Tnmd on cell adhesion of PDL-h<i>TERT</i> cells to col I-coated culture dishes. The fluorescence intensity was measured using a confocal laser scanner and the percent cell adhesion was calculated.</p

Expression of Tnmd in the mouse periodontal ligament.

<p>(<b>A</b>) Immunohistochemical detection of Tnmd in the incisors. Tnmd localization was examined by IHC in mandible incisors obtained from 4-week-old mice. (<b>B</b>) Immunohistochemical detection of Tnmd in molars. The temporal expression of Tnmd was examined by IHC in maxilla molars obtained from 2, 3, and 4-week-old mice. e: enamel organ; d: dentin; g: gingiva; pul: pulp; pdl: periodontal ligament; b: bone. The dotted line indicates the area of periodontal ligament. Arrow indicates the region of positive signal. Scale bar = 500 µm.</p