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. 2023 Oct 26;11:56. doi: 10.1038/s41413-023-00296-3

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© The Author(s) 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. 2 — Loss of Trim21 enhances osteoblast activity and favors bone formation. a, b Representative immunoblotting analysis (a) and quantification of Runx2, Osterix, and Trim21 in MC3T3-E1 cells (b) treated with osteogenic medium for 0, 4, and 7 days. c Quantitative RT‒PCR analysis of osteogenic biomarker genes (Osterix, Runx2, and Trim21) in OBs with osteogenic induction. d, e Alizarin Red S (upper panel) and ALP (lower panel) staining of primary osteoblasts (OBs) after induction with osteogenic medium for different times (d). The percentage of Alizarin Red S- (n ≥ 3) and ALP- (n ≥ 3) stained area (e). f Quantitative RT‒PCR detection of osteogenic biomarker genes (Runx2, Osterix, OCN, OPG, and RANKL) in OBs derived from Trim21^−/− and Trim21^+/+ mice upon osteogenic induction for 7 days. g, h Representative immunoblotting analysis (g) and quantification of Runx2 and Osterix in OBs (h) after osteogenic induction for 0, 4, and 7 days. i Representative micro-CT images of calvarial bone defects in 2-month-old Trim21^+/+ and Trim21^−/− mice after surgical induction for 1 month (left panel). Quantitative measurements of bone volume per tissue volume (BV/TV) and bone defect diameter (right panel). All bar graphs are presented as the mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.000 1; n.s. not significant by Student’s t test