Sequence of primers used in real-time RT-PCR.

<p>Sequence of primers used in real-time RT-PCR.</p

Age effect on the relative overexpression levels of <i>OA</i> mRNA in femurs of female <i>OA</i>-Tg mice.

<p>Total RNA was isolated from femurs of female <i>OA</i>-Tg mice of age of 4-, 8-, and 15.3-week-old and corresponding WT littermates (3–5 mice per group) and cDNA was prepared as described in Methods. The relative level of <i>OA</i> mRNA, determined by real-time RT-PCR and normalized against respective β-actin mRNA level), is reported as fold of that in corresponding age-matched WT osteoclasts (indicated by the dashed line) and shown as mean ± SEM (n = 3–5).</p

Effects of siRNA-mediated <i>OA</i> suppression on average cell size (A), <i>in vitro</i> bone resorption activity (B), and expression of osteoclastic genes (C) in RAW264.7 cell-derived osteoclast-like cells.

<p>The dosage of <i>OA</i> siRNAs (29 pM) used in this experiment suppressed OA expression in RAW264.7 cells by greater than 70% (data not shown). RAW264.7 cells were treated with <i>OA</i> siRNAs or control siRNA in the presence of RANKL for 5 days. A shows the relative size of the derived TRAP positive, multinucleated osteoclast-like cells. Top is a representative photomicrograph of the derived osteoclast-like cells, and bottom summarizes the relative size (in relative percentage of the control siRNA-treated cells). B shows the bone resorption activity of the derived osteoclast-like cells determined by an <i>in vitro</i> resorption pit formation assay; and C summarizes the effects of OA siRNA on the relative expression levels of <i>MMP9</i>, <i>CALCR</i>, and <i>NFATc1</i> mRNA (determined by real-time RT-PCR and normalized by the respective expression level of β-actin). Results are shown as percentage of respective control siRNA-treated RAW264.7 cell-derived osteoclast-like cells and in mean ± SEM (n = 3 or 4 for each parameter). The dashed line represents the 100% of the control siRNA-treated controls.</p

Comparison of static histomorphometric trabecular bone parameters at the secondary spongiosa of 4-week-old female <i>OA</i>-Tg mice with age- and sex-matched WT littermates (mean ± SEM).^*

*<p>Measurments were performed at a site that was 300 µm away from the growth plate.</p>**<p>N.S. = Not significant.</p>***<p>% (BV/TV), trabecular area per total tissue area in percentage; Total OC#, total number of TRAP positive multinucleated osteoclasts; <i>OC#.PM</i>, number of TRAP positive osteoclasts per bone surface length; TRAP.PM, TRAP-stained bone surface per total tissue area; Tb.N, trabecular number; Tb.Th, trabecular thickness; and Tb.Sp, trabecular spacing.</p

Schematic representation of the pGL3-TRAP-1B/C-<i>OA</i> expression plasmid.

<p>The pGL3-TRAP-1B/C-<i>OA</i> expression plasmid was generated by cloning the full-length mouse <i>OA</i> cDNA into the Hind3/XbaI restriction site of the pGL3-basic vector. The TRAP-1B/C promoter was then cloned into the Kpn1/Hind3 restriction sites.</p

Comparison of μ-CT bone parameters at the secondary spongiosa of 4-week-old female <i>OA</i>-Tg mice with those of 4-week-old female WT littermates.

<p>Top panels show the three-dimensional reconstruction of bone structure by μ-CT at the secondary spongiosa of two representative <i>OA</i>-Tg mice (right) and two WT littermates (panel). Bottom summarizes and compares the various μ-CT bone parameters of a group of four <i>OA</i>-Tg mice with a group of four WT littermates.</p

Targeted overexpression of <i>OA</i> in cells of osteoclastic lineage increased circulating levels of c-telopeptide of type I collagen.

<p>Plasma c-telopeptide levels of 8-week-old male young adult <i>OA</i>-Tg mice (n = 17) and WT littermates (n = 12) were measured with a commercial ELISA assay, and results are shown as mean ± SEM.</p

Comparison of bone and pQCT parameters of 8 weeks old <i>OA</i> transgenic (Tg) mice with targeted <i>OA</i> overexpression in osteoclastic cells to those of 8 weeks old sex-matched WT littermates (mean±SEM).

*<p>Metaphysis parameters were measured at 22% in length down from the distal end of the femur;</p>#<p>Mid-diaphysis parameters were measured at the mid-shaft of the femur.</p

Effects of targeted overexpression of <i>OA</i> in osteoclastic cells on plasma levels of biomarkers of bone formation <i>in vivo</i>.

<p>In A, plasma levels of osteocalcin of female <i>OA</i>-Tg mice and WT littermates of 8 or 15.3 weeks of age were measured with a commercial ELISA kit. Results are shown as mean ± SEM with the indicated the number of mice per group. In B, plasma levels of pro-collagen type I N-terminal peptide (PINP) of both male and female 15.3-week-old <i>OA</i>-Tg mice and corresponding age- and sex-matched WT littermates were measured with a commercial ELISA kit. Results are shown as mean ± SEM with the indicated number of mice per group.</p

Effects of targeted overexpression of <i>OA</i> overexpression in osteoclastic cells on histomorphometric bone formation parameters in 8-week-old male <i>OA</i>-Tg and also in 15.3-week-old female <i>OA</i>-Tg mice (mean±SEM).

<p>BS, bone surface; TLS, tetracycline labeling surface; MAR, mineralization apposition rate; BFR, bone formation rate; E.BS, endosteal bone surface; E.TLS, endosteal tetracycline labeling surface; E.MAR, endosteal mineralization apposition rate; E.BFR, endosteal bone formation rate.</p>*<p>Dynamic bone formation parameters were performed on longitudinal sections of femurs of 7 WT littermates and 13 <i>OA</i>-Tg mice at the cortical bone site of the mid-shaft, starting from 1.2 mm from the lowest point, 2 grids under a 10× microscope lens.</p>#<p>Dynamic bone formation parameters were performed on the endosteal surface of femurs of 8 WT littermates and 11 <i>OA</i>-Tg mice.</p