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. 2022 Jun 27;13:3684. doi: 10.1038/s41467-022-31356-7

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© The Author(s) 2022

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 — MLO-Y4 and MLO-A5 osteocytes carried with non-targeted control shRNAs (shCtrl) or Ciita shRNAs (shCiita) were cultured with or without 2DDR for 2 days, given fresh medium, and cultured for another 2 days, and then the cells and the CM were collected. The osteoblast precursor MC3T3-E1 and the osteoclast precursors Raw264.7 cells or primary monocytes were cultured with or without the osteocyte CM. After culturing, the cells were subjected to Alizarin red-S staining to assess osteoblast differentiation, to TRAP staining to assess osteoclast differentiation, or to real-time PCR to assess the differentiation-associated gene expression. a–f Shown are summarized data for Sost mRNAs (a), Alizarin red-S staining (b), the relative expression of osteoblast differentiation–associated genes Alp, Col1a1, and Bglap (c), Tnfsf11 mRNA (d), the numbers of multinuclear (≥3) TRAP⁺ cells (e), and the relative expression of osteoclast differentiation–associated genes Trap, Calcr, and Ctsk (f). Data are mean ± SD (n = 3 biological replicates). g–j Osteocytes isolated from WT or osteocyte-specific Ciita-KO mice (n = 5 mice/group) were cultured with or without 1 mM 2DDR for 2 days for collecting the cells and the osteocyte CM. MC3T3-E1 or Raw264.7 cells cultured with osteocyte CM were subjected to Alizarin red-S staining, TRAP staining, or real-time PCR. Shown are summarized data for Sost mRNA levels in osteocytes (g), Alizarin red-S staining (h), Tnfsf11 mRNA levels in osteocytes (i), and numbers of multinuclear (≥3) TRAP⁺ cells (j, left). Box plots (g, i) indicate median, 25^th and 75^th percentiles, and minima and maxima of the distribution. Data are presented as mean ± SD. (j, right) Primary osteocytes wild-type or carried with Ciita siRNAs (siCiita) were cultured in the same fashion as in (a) to collect the CM. The numbers of multinuclear (≥3) TRAP⁺ cells were counted in the cultures of primary monocytes with or without the osteocyte CM. Data are means ± SD. P values were determined using one-way ANOVA with Tukey’s multiple comparisons test.