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. 2019 Jun 26;10:2795. doi: 10.1038/s41467-019-10677-0

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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. 1 — TRAF3 cKO mice have early onset osteoporosis. a, b Representative µCT images and bone volume (BV/TV) values in a tibiae and b L1 vertebrae from litters of 3-, 9-, and 15-month (m)-old WT and TRAF3 conditional knockout (cKO) mice, including both males and females, as listed in the figure. Mean ± SD (3-m-old WT (n = 7) and cKO (n = 8), 9-m-old WT (n = 10) and cKO (n = 11), 15-m-old WT (n = 11) and cKO (n = 8) biologically independent mice; *p < 0.05, **p < 0.01). Scale bar, 1 mm. c Western blot (WB) of TRAF3/GAPDH in lysates of femoral metaphyses (F; upper panel) and vertebrae (V; lower panel) from 3- and 15-m-old WT and cKO mice. d Representative H&E-stained sections of proximal tibiae of 3- and 9-m-old WT and TRAF3 cKO mice. Scale bar, 400 μm. e Mineralizing surface (MS/BS), mineral apposition rate (MAR), and bone formation rate (BFR) analyzed in calcein double-labeled plastic sections of L1 vertebrae from the mice in (a). Mean ± SD (3-m-old WT (n = 5) and cKO (n = 6), 9-m-old WT (n = 7) and cKO (n = 7), 15-m-old WT (n = 5) and cKO (n = 6) biologically independent samples; *p < 0.05, **p < 0.01). f Representative H&E-stained images of vertebral sections from 9-m-old WT and cKO mice showing osteoblasts (yellow arrows) on the trabecular bone (Tr.B) surfaces, and histomorphometric analysis of osteoblast surfaces (OB.S/BS) from the mice in (a). Mean ± SD (3-m group: n = 4, other groups: n = 6 biologically independent samples; **p < 0.01). Scale bar, 100 μm. g Serum osteocalcin values tested by ELISA from the 3-, 9-, and 15-m-old WT and cKO mice in (a). Mean ± SD (n = 8 biologically independent samples; **p < 0.01). h Representative images of TRAP-stained vertebral sections from 9-m-old WT and cKO mice, and histomorphometric analysis of osteoclast numbers (Oc.N) and surfaces (Oc.S) from the mice in (a). Mean ± SD (3-m group: n = 5, other groups: n = 6 biologically independent samples; *p < 0.05). Scale bar, 100 μm. i Serum TRACP-5b levels tested by ELISA from the mice in (a). Mean ± SD (n = 8 biologically independent samples; *p < 0.05, **p < 0.01). All analyses done using one-way ANOVA with Tukey’s post-hoc test