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. 2022 Mar 9;10:27. doi: 10.1038/s41413-022-00201-4

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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. 1 — Expression of EP2 and EP4 in subchondral bone osteoclasts and the effects of PGE2 on osteoclasts with EP2 and EP4 deletion. a qRT-PCR analysis of the mRNA expression levels of EP1, EP2, EP3, EP4, and Trap in osteoclasts differentiated from BMMs exposed to 10 ng·mL⁻¹ M-CSF and 50 ng·mL⁻¹ RANKL for 5–6 days. Error bars are the mean ± s.d. n = 3. *P < 0.05, **P < 0.01 and ***P < 0.001, ns, not significant by unpaired two-tailed Student’s t test. b Representative images of the protein levels of EP2 and EP4 in mouse tibial subchondral bone tissue 2 weeks after sham or ACLT surgery. The experiments were performed with three biological replicates. c Representative images of IHF staining of EP2 or EP4 (green) and TRAP (red) in subchondral bone 2 weeks post-sham or ACLT surgery (left) and quantitative analysis (right). The white arrows indicate TRAP-positive osteoclasts expressing EP2 or EP4. Error bars are the mean ± s.d. *P < 0.05, ns, not significant by unpaired two-tailed Student’s t test. n = 4 for per group. Scale bars, 20 μm. d EP2 deletion inhibits PGE2-induced migration and osteoclast differentiation. BMMs from the EP2^WT and littermate EP2^KO mice were used to generate osteoclasts by stimulation with 10 ng·mL⁻¹ M-CSF and 50 ng·mL⁻¹ RANKL and incubation with 100 nmol·L⁻¹ PGE2. Representative image of cells from the Transwell migration assay (Transwell) and osteoclast differentiation assay (TRAP staining) (top) and the corresponding quantitative analysis (bottom). Error bars are the mean ± s.d. n = 3. Two-way ANOVA followed by Tukey’s t tests. Scale bars, 50 μm. e EP2 deletion inhibits PGE2-induced migration and osteoclast differentiation. BMMs from the WT (EP4^fl/fl) and littermate EP4^fl/fl: LysM-cre (EP4^LysM) mice were used to generate osteoclasts by stimulation with 10 ng·mL⁻¹ M-CSF and 50 ng·mL⁻¹ RANKL (5–6 days) and incubation with 100 nmol·L⁻¹ PGE2. Representative image of cells from the Transwell migration assay (Transwell) and osteoclast differentiation assay (TRAP staining) (top) and the corresponding quantitative analysis (bottom). Error bars are the mean ± s.d. n = 3. Two-way ANOVA followed by Tukey’s t tests. Scale bars, 50 μm