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. 2022 Mar 8;10:25. doi: 10.1038/s41413-022-00204-1

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© The Author(s) 2022, corrected publication 2023

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 — Senescent preosteoblasts present elevated mTORC1 activity. a Representative microcomputed tomography (μCT) images of femurs of 5-month-old (mo) and 18-month-old (mo) ΔRaptor mice compared with those of the littermate controls. Scale bar: 500 μm. b Quantification of trabecular bone volume per total volume (Tb.BV/TV), trabecular number (Tb.N), trabecular separation (Tb.Sp) and trabecular thickness (Tb.Th). Representative images of double immunostaining of Osx plus p16 (c) and Osx plus pS6 (Ser235/236) (d) in the bones of the mice in a. Single Osx staining served as a negative control (NC) for double staining of Osx plus p16 in c. Scale bars: 100 μm. e The murine preosteoblast cell line MC3T3-E1 was exposed to senescence induction by reactive oxygen species (ROS) or treated with vehicle (Veh) and stained for the expression of senescence-associated β-galactosidase (SA-β-gal) 3 days later. Scale bar: 50 μm. Quantification of the proportion of SA-β-gal⁺ preosteoblasts (pOBs) in each population is shown. f Representative confocal images of immunostaining of EdU (red) in the cells in e and quantitative analysis of EdU⁺ pOBs relative to total cells (%). Scale bar, 100 μm. g Western blot analysis of senescence marker (p16 and p53) expression and mTORC1 activity [pS6(Ser235/236)] in the cells in e. Data are shown as the mean ± SD. The numbers of samples (n) are indicated in each figure panel. P values were determined by two-tailed Student’s t test for single comparisons