Predominant nonsynonymous env mutations.

<p>Viral cDNAs encoding Env were amplified from plasma RNAs obtained at 7-9 months (R10-001, R10-004, and R06-029) or 12 months (other animals) and subjected to sequencing analysis. Amino acid substitutions are shown. The asterisk (*) represents a deletion and the double asterisk (**) represents coexistence of multiple deletion patterns. </p

Binding properties of IgGs to SIV antigens.

<p>The non-NAb cocktail, ten non-NAb IgG lots derived from ten macaques, and CAb were subjected to immunoblotting (ZeptoMetrix). A representative result from two experiments is shown.</p

SIV antigen-specific CD8⁺ T-cell responses.

<p>SIV Gag-, Pol-, Vif-, Vpx/Vpr-, Tat-, Rev-, Nef-, and Env-specific CD8⁺ T-cell responses were measured by detection of antigen-specific IFN-γ induction using PBMCs at weeks 26-30 post-challenge.</p

Binding properties of IgGs to SIV virions.

<p>Polyclonal IgGs purified from macaque plasma were subjected to whole virus ELISA using purified SIV_mac239 virions as the antigen. Results on ten IgG lots derived from ten macaques without detectable neutralizing activity (non-NAbs; black lines), five with neutralizing activity (NAbs; red), and a control IgG (CAb; green) are shown in the left panel. Results on the non-NAb cocktail and three non-NAb lots composing the cocktail are in the right. The dotted line represents background absorbance. Time points of plasma sampling are shown in parentheses following the macaque IDs. A representative result, means and SDs of duplicate samples, from two experiments is shown.</p

ADCVI activity of the non-NAb cocktail and non-NAb IgG lots.

<p>The reduction in SIV p27 concentration in the supernatant from SIV-infected cell culture with non-NAbs compared to that without antibodies is shown. A representative result, means of duplicate samples, from two experiments is shown.</p

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shRNA-mediated knockdown of Jak1 or Jak2 also suppressed expression of RANKL in osteoblasts.

<p><b>(A)</b> Immunoblot images of adenovirus-mediated knockdown of murine Jak1 or Jak2. <b>(B, C)</b> Effects of shJak1_#1, #2, shJak2_#1 or #2 on osteoclast formation in co-cultures of osteoblasts and bone marrow cells treated with 1,25D₃ and PGE₂ <b>(B)</b> or in bone marrow macrophage cultures treated with RANKL and M-CSF <b>(C)</b>. <b>(D, E)</b> Effects of shJak1_#1, #2, shJak2_#1 or #2 on expression of RANKL mRNAs <b>(D)</b> and proteins <b>(E)</b> in osteoblasts. Osteoblasts were transfected with shCtrl (lane 1, lane 2), shJak1_#1 (lane 3), shJak1_#2 (lane 4), shJak2_#1 (lane 5) or shJak2_#2 (lane 6) and treated with (lane 2–6) or without (lane 1) 1,25D₃ and PGE₂ for 24 h. In <b>B–D</b>: error bars, s.e. (n = 3–4). **<i>P</i> < 0.01, Student's <i>t</i> test. † †<i>P</i> < 0.01, Dunnett’s multiple comparisons test (vs shCtrl with 1,25D₃ and PGE₂). Original immunoblot images are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181126#pone.0181126.s001" target="_blank">S1 Fig</a>.</p

Baricitinib inhibits osteoclastogenesis by suppressing RANKL expression in osteoblasts.

<p><b>(A)</b> The dose-response curve of viability of osteoblasts treated with baricitinib for 24 h. 100% was determined as the cell numbers when treated with vehicle (DMSO). <b>(B)</b> Effects of 2.5 μM baricitinib on expression of <i>RANKL</i>, <i>M-CSF</i>, and <i>OPG</i> mRNAs in osteoblasts in the presence of 1,25D₃ and PGE₂. <b>(C, D)</b> 200 ng ml^-1 GST-RANKL, but not 50 ng ml^-1 M-CSF, completely cancelled effects of 2.5 μM baricitinib on osteoclast formation in co-cultures. Micrographs show TRAP staining of osteoclasts. Scale bar, 500 μm. In <b>A, B,</b> and <b>D</b>: error bars, s.e. (n = 6 (A), n = 3–4 (B, D)). **<i>P</i> < 0.01, Student’s <i>t</i> test. † †<i>P</i> < 0.01, n.s. not significant, Dunnett’s multiple comparisons test (vs vehicle <b>(A)</b> or vehicle with 1,25D₃ and PGE₂ <b>(D)</b>).</p

Histomorphometric analysis of the protective effects of W9 on alveolar bone loss in <i>OPG</i>^<i>-/-</i> mice.

<p>Histomorphometric analysis of the protective effects of W9 on alveolar bone loss in <i>OPG</i>^<i>-/-</i> mice.</p

Effects of W9 and risedronate administration on Wnt/β-catenin signaling of alveolar bone in <i>OPG</i>^<i>-/-</i> mice.

<p>(A) Histological analysis of the interradicular septum of the first molar (M1) in maxillae from WT and <i>OPG</i>^–/–mice treated with and without W9 or risedronate. β-catenin staining of WT and <i>OPG</i>^–/–mice. β-catenin-positive cells in nuclei (brown) were observed in the M1 interradicular septum in alveolar bone areas. (B) Sclerostin and TRAP double staining of WT and <i>OPG</i>^–/–mice. Sclerostin-positive osteocytes (brown) were observed in the M1 interradicular septum in alveolar bone areas. Sclerostin-positive osteocytes are indicated by black arrows. (C) The number of sclerostin-positive cells/bone area (N/mm²) was determined in the M1 interradicular septum (<i>n</i> = 5). Data are expressed as the mean ± SD. *: p<0.05. Scale bar, 50 μm.</p