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. 2019 May 13;7:14. doi: 10.1038/s41413-019-0054-y

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© The Author(s) 2019

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. 3 — HIF-2α increases osteoblast-mediated osteoclastogenesis. a–c Primary calvarial preosteoblasts were infected with Ad-C or Ad-Hif-2α. qRT-PCR analysis of Rankl, Opg (left panel), and the Rankl/Opg ratio (right panel) (n = 3; a). ELISA analysis of secreted RANKL in culture media (n = 4; b) and detection of RANKL by immunofluorescence microscopy (scale bar: 100 μm; c). d Determination of Hif-2α, Rankl, and Opg expression following siRNA-mediated knockdown of HIF-2α in L-AA/β-GP-induced osteoblasts (n = 3). e RANKL immunostaining in bone obtained from Hif-2α^+/+ and Hif-2α^+/− mice. Scale bar: 100 μm. Rankl-positive osteoblasts were counted in the epiphyseal bone compartment. f Primary calvarial preosteoblasts infected with Ad-Hif-2α and BMMs were mixed and cocultured on glass covers, and TRAP staining was performed. g Osteoclasts derived from WT mice and osteoblasts from HIF-2α^+/+ or HIF-2α^+/− mice in the absence or presence of BMP-2 (100 ng/ml) were cocultured, and TRAP staining was performed (scale bar: 100 μm). h A ChIP assay of Ad-Hif-2α-infected osteoblasts was performed with anti-HIF-2α antibody and a primer pair designed to span the putative HIF-2α binding regions within the promoter of Rankl. Values are presented as the mean ± SEM (*P < 0.05, **P < 0.01, and ***P < 0.005; NS not significant)