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. 2021 Oct 20;12(11):971. doi: 10.1038/s41419-021-04275-6

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 6 — A M-CSF (40 ng/ml) was added to monocyte culture for three days. OCPs that were transiently transfected with B7–H3-specific siRNA or control siRNA (40 nM) were induced to differentiate using M-CSF (40 ng/ml) and RANKL (80 ng/ml) for seven days. B The M-CSF (40 ng/ml) was added in monocyte culture for three days, then the cells were transfected with human pCMV3–B7–H3 ORF-expression plasmid or pCMV3-negative control vector (2 μg) for one day. The expression of B7–H3, IDO, phospho-STAT1 (PY701), and STAT1 protein during osteoclast differentiation was analyzed by Western blot. C OCPs that were transiently transfected with B7–H3-specific siRNA (20 nM), IDO-specific siRNA (60 nM), control siRNA (80 nM), or both B7–H3- and IDO-specific siRNA were induced to differentiate using M-CSF (40 ng/ml) and RANKL (80 ng/ml) for seven days. The cells were stained for TRAP expression and TRAP-positive multinucleated cells were counted as osteoclasts. Actin rings in osteoclasts stained with FITC-phalloidin (scale bar, 200 μm). D The expression of mature osteoclast markers, CTSK, TRAP, DC-STAMP, and ITGB3, was analyzed using RT-qPCR. The mRNA levels were normalized relative to GAPDH expression. E The expression of B7–H3, IDO, and NFATc1 protein was analyzed via Western blot. F OCPs that were transiently transfected with B7–H3-specific siRNA or control siRNA (20 nM) were induced to differentiate using M-CSF (40 ng/ml) and RANKL (80 ng/ml) in the presence of anti-IFN-αR2, -IFN-γ, or -IL-27 for three days. The expression of B7–H3, IDO, phospho-STAT1 (PY701), and STAT1 protein was analyzed via Western blot. G Monocytes were treated with recombinant IFN-β (1 ng/ml) or distilled water for one or two days. Whole-cell lysates were immunoblotted with IDO, phospho-STAT1 (PY701), and STAT1 antibodies. H OCPs that were transiently transfected with IDO-specific siRNA or control siRNA (60 nM) were induced to differentiate using M-CSF (40 ng/ml) and RANKL (80 ng/ml) in the presence of recombinant IFN-β (1 ng/ml) or distilled water for seven days. The cells were stained for TRAP expression (scale bar, 200 μm). I At days 1 and 3, whole-cell lysates were immunoblotted with IDO, phospho-STAT1 (PY701), and STAT1 antibodies.