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. 2021 Aug 25;12:2040622321995685. doi: 10.1177/2040622321995685

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© The Author(s), 2021

This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 License (https://creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage).

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Figure 6. — Melatonin stimulates osteogenic differentiation of BMSCs via the HGF/PTEN/Wnt/β-catenin axis in BMSCs.

(a) mRNA expression of HGF and PTEN in BMSCs treated with Met-100 μmol/l or in combination with oe-PTEN determined by RT-qPCR. (b) Activity of TOPflash in BMSCs treated with Met-100 μmol/l or in combination with oe-PTEN detected by dual-luciferase reporter. (c) Western blot analysis of HGF, PTEN and β-catenin protein expression in BMSCs treated with Met-100 μmol/l or in combination with oe-PTEN. (d) mRNA expression of HGF and PTEN in BMSCs treated with Met-100 μmol/l or in combination with DKK1 determined by RT-qPCR. (e) Activity of TOPflash in BMSCs treated with Met-100 μmol/l or in combination with DKK1 detected by dual-luciferase reporter. (f) Western blot analysis of HGF, PTEN and β-catenin protein expression in BMSCs treated with Met-100 μmol/l or in combination with DKK1. (g) Proliferation of BMSCs treated with Met-100 μmol/l or in combination with DKK1 measured by CCK-8 assay. (h) Mineralization of BMSCs treated with Met-100 μmol/l or in combination with DKK1 evaluated by ARS staining. (i) Calcification of BMSCs treated with Met-100 μmol/l or in combination with DKK1 evaluated by ALP activity and staining. (j) Western blot analysis of BMP4, Runx2, Sp7, OCN, OPN and BMP2 protein expression in BMSCs treated with Met-100 μmol/l or in combination with DKK1. ^**p < 0.01, indicates statistical significance. Data (mean ± standard deviation) among multiple groups were compared by one-way ANOVA with Tukey’s test while those at different time points were compared by two-way ANOVA with Bonferroni’s correction. Cell experiments were conducted in triplicate.

ALP, alkaline phosphatase; ANOVA, analysis of variance; ARS, alizarin red S; BMSCs, bone marrow mesenchymal stem cells; CCK, cell-counting kit; Met, melatonin; mRNA, messenger ribonucleic acid; oe-, overexpression; OVX, ovariectomy; RT-qPCR, real-time qualitative polymerase chain reaction.