Housing Conditions.

<p>Illustration of the tail ring (A), and steel wires with tape (B) as applied to the tail for the suspension studies. Also shown are the three different housing conditions; normal housing (C), non suspended housing (D), and suspended housing (E) using terminated immobile mice to show angles of suspension as active mice in non suspended housing (F) and suspended (G) are difficult to photograph. The food blocks were clamped (black boxes) to allow access but to prevent movement of food block for resting of hindlimbs.</p

Corticosterone levels for 3 different housing conditions and 2 genotypes.

<p>Ctrl: Control, HET: Dmp1 Cre x β-catenin heterozygotes, NH: Normal Housing, NS: Non Suspended, SUS: Suspended. Number of animals per group = 5–8. *: represents a significant difference between two groups (p<0.05). One-way Analysis of Variance was used to assess the differences between the groups followed by pairwise post hoc tests.</p

Body weight change for 3 different housing conditions and 2 genotypes.

<p>Ctrl: Control, HET: Dmp1 Cre x β-catenin heterozygotes, NH: Normal Housing, NS: Non Suspended, SUS: Suspended. Number of animals per group = 5–8. *: represents a significant difference between two groups (p<0.05). One-way Analysis of Variance was used to assess the differences between the groups followed by pairwise post hoc tests.</p

Osteoclast activity as measured by TRAP staining after sacrifice at 19 weeks.

<p>Osteoclast activity as measured by TRAP staining after sacrifice at 19 weeks.</p

Bone phenotype of Control mice (Ctrl) as compared to β-catenin conditional knock-out heterozygous mice (cKO HET) at 14 weeks (<i>in vivo</i>) and 19 weeks (<i>ex vivo</i>).

<p>Bone phenotype of Control mice (Ctrl) as compared to β-catenin conditional knock-out heterozygous mice (cKO HET) at 14 weeks (<i>in vivo</i>) and 19 weeks (<i>ex vivo</i>).</p

Trabecular and cortical bone regions analyzed by microCT.

<p>Trabecular bone microarchitecture was analyzed below the growth plate. 1.2mm was analyzed, which represents 63 slices <i>in vivo</i> (19 μm) and 120 slices <i>ex vivo</i> (10 μm voxel size). Cortical bone microarchitecture was analyzed below the growth plate (1mm analyzed representing 53 slices <i>in vivo</i> and 100 slices <i>ex vivo</i>) and above the tibia-fibula junction (0.5mm analyzed representing 26 sections <i>in vivo</i> and 50 sections -<i>ex vivo</i>).</p

Effects of environment and suspension on 19 week old control and cKO HET male mice.

<p>Cortical bone mineral density (BMD), bone volume (BV/TV) and cortical thickness (Ct.Th) were analyzed at the diaphysis and at the metaphysis as shown in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158381#pone.0158381.g002" target="_blank">Fig 2</a>. Trabecular bone mineral density (BMD), bone volume (BV/TV), number (Tb.N), thickness (Tb.Th) and spacing (Tb.Sp) were analyzed at the metaphysis. *significant difference between NH and NS and between NS and SUS (p<0.05). One-way Analysis of Variance was used to assess the differences between the groups followed by pairwise post hoc tests. Number of animals per group = 5–7.</p

UV emission peak intensity ratios in osteocyte and surrounding matrix ROIs for the three different groups.

<p><b>A</b>: Tyrosine/Collagen ratio in osteocyte ROI. <b>B</b>: Tyrosine/Collagen ratio in matrix ROI. <b>C</b>: Tryptophan/Collagen ratio in osteocyte ROI. <b>D</b>: Tryptophan/Collagen ratio in matrix ROI. <b>E</b>: Tyrosine/Collagen ratio in osteocyte ROI. <b>F</b>: Tyrosine/Collagen ratio in matrix ROI. shows a significant difference between the groups. Ratios are expressed as mean ± standard deviation (sd).</p

UV spectroscopy results from osteocyte and surrounding matrix.

<p><b>A</b>: Transmission microscopy image showing ROIs, white scale bar 10 µm. <b>B</b>: UV fluorescence spectrum originating from osteocyte ROI pixel “o”. <b>C</b>: UV fluorescence spectrum originating from matrix ROI pixel “m”. <b>D</b>: Deconvolution of spectrum “m” into three Gaussians corresponding to tyrosine (a1), tryptophan (a2), and collagen (a3).</p

Tyrosine/Collagen and Tryptophan/Collagen ratios on osteocyte and surrounding matrix for the A25 group.

<p><b>A</b>: Comparison between osteocyte and matrix for the Tyrosine/Collagen ratio. <b>B</b>: Comparison between osteocyte and matrix for the Tryptophan/Collagen ratio. shows a significant difference between the ratios (p<0.05). Ratios are expressed as mean ± standard deviation (sd).</p