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. 2020 Nov 11;11:5704. doi: 10.1038/s41467-020-19555-6

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© The Author(s) 2020

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. 2 — a Femurs from 16-week-old Nf1^fl/fl, Nf1^fl/fl;Mekk2^−/−, Nf1^fl/fl;Dmp1-Cre, and Nf1^fl/fl;Mekk2^−/−;Dmp1-Cre mice were analyzed by μCT. b Quantitative parameters are porous volume, porosity (porous volume/total volume), and porous surface from 16-week-old Nf1^fl/fl (n = 5), Nf1^fl/fl;Mekk2^−/− (n = 8), Nf1^fl/fl;Dmp1-Cre (n = 8), and Nf1^fl/fl;Mekk2^−/−;Dmp1-Cre (n = 10) mice. Mean ± s.d., one-way ANOVA with Tukey’s multiple comparison test. c Representative μCT 3D-reconstuction images of trabecular bone in the distal femur metaphysis. d Quantitative parameters include trabecular bone volume/total volume (BV/TV), thickness (Tb.Th), trabecular number (Tb.N), and spacing (Tb.sp) in 16-week-old Nf1^fl/fl (n = 8), Nf1^fl/fl;Mekk2^−/− (n = 6), Nf1^fl/fl;Dmp1-Cre (n = 9), and Nf1^fl/fl;Mekk2^−/−;Dmp1-Cre (n = 8) mice. mean ± s.d., one-way ANOVA with Tukey’s multiple comparison test. e μCT scans of mouse skulls at 16 weeks Nf1^fl/fl, Nf1^fl/fl;Mekk2^−/−, Nf1^fl/fl;Dmp1-Cre, and Nf1^fl/fl;Mekk2^−/−;Dmp1-Cre mice. f Serum levels of P1NP, CTX, FGF23, PTH, 25(OH) vitamin D, and phosphate in 16-week-old mice. Data are represented as box plots with the middle line representing the median, the box representing the 95% confidence interval of the median, and the whiskers representing the range. Each dot represents a separate mouse. g Representative images and quantification of immunostaining for p-MEKK2 (green), p-MEK1 (magenta), and p-ERK (red) in trabecular (left panels) and cortical bone (right panels) from 16-week-old Nf1^fl/fl, Nf1^fl/fl;Dmp1-Cre, and Nf1^fl/fl;Mekk2^−/−;Dmp1-Cre mice. Nuclei are counterstained with DAPI (blue) and the scale bar indicates 100 µm. Three independent fields were examined per mouse (n = 3 mice per group). mean ± s.d., one-way ANOVA with Tukey’s multiple comparison test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.