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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. 4 — Osteoblast-specific depletion of HIF-2α increases bone mass. a Osteoblast-specific depletion of HIF-2α in Hif-2α^fl/fl and Hif-2α^fl/fl;Col1a1-Cre mice was determined by immunohistochemistry with anti-HIF-2α antibody. Scale bar: 10 μm b, c Analyses of femoral trabecular bones from 4-month-old Hif-2α^fl/fl and Hif-2α^fl/fl;Col1a1-Cre mice. Representative images of µCT reconstructions of trabecular bones (b) and H&E and TRAP staining (c). BV/TV, Tb.Th, Tb.Sp, and Tb.N were assessed based on the µCT measurements (n = 8; b), and BV/TV, N.Ob/B.Pm, Ob.S/BS, N.Oc/B.Pm, and Oc.S/BS were determined from the bone histomorphometric analysis of the metaphyseal regions of femurs (n = 8; c). Scale bar: 100 μm. d, e Quantitative µCT analysis of femoral trabecular bones (n = 8; d) and ELISA-based measurement of the serum concentrations of OCN (n = 5; e) and CTX-1 (n = 9; e) in OVX- or sham-operated 3-month-old Hif-2α^fl/fl and Hif-2α^fl/fl;Col1a1-Cre mice. f, g Osteoblast differentiation was validated in primary calvarial preosteoblasts from Hif-2α^fl/fl mice infected with Ad-C or Ad-Cre in the presence of differentiation medium. Osteoblast differentiation was examined by ALP and ARS staining (f), and its corresponding gene expression was determined by qRT-PCR (n = 4; g). Values are presented as the mean ± SEM (*P < 0.05; **P < 0.01, and ***P < 0.005). The effects of OVX and osteoblast-specific deletion of Hif-2α (cΚΟ) as well as their interaction in mice were analyzed by two-way ANOVA (d BV/TV: interaction = 0.034 1, OVX < 0.000 1, cΚΟ < 0.000 1; e OCN: interaction < 0.000 1, OVX < 0.000 1, cΚΟ < 0.000 1; e CTX-1: interaction = 0.041 4, OVX < 0.000 1, cΚΟ = 0.046 1)