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. 2016 May 12;7(5):e2221. doi: 10.1038/cddis.2016.112

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Copyright © 2016 Macmillan Publishers Limited

Cell Death and Disease is an open-access journal published by Nature Publishing Group. This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

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HDAC1 overexpression blocked CMS-induced osteogenic differentiation in human BMSCs. (a) Scheme of primers' location in the 5′-flank promoter region of JAG1 gene. The transcriptional start site (TSS) is indicated as +1. (b) Western blotting analysis revealed a increase in acetylation of histone H3 (H3) in BMSCs after treatment with 10% CMS for 3 weeks compared with static control cells. (c) ChIP-qPCR analysis of H3 acetylation modification in different JAG1 promoter regions in BMSCs after treatment with 10% CMS for 3 weeks compared with static control cells. (d) ChIP-qPCR assay on GAPDH and JAG1 promoters. ChIP analysis revealed that there was a significant increase in histone H3 acetylation at JAG1 promoters after siRNA-HDAC1 treatment in BMSCs. (e) The efficiency of HDAC1 overexpression was confirmed by comparison to a empty vector. (f) qRT-PCR analysis of osteogenic differentiation markers ALP, COL1a1, and OCN. (g) ALP staining (including quantitative analysis) and (h) Alizarin red staining (including quantitative analysis) in BMSCs after transfected with HDAC1 overexpression or its corresponding negative control under 10% CMS for 3 weeks. (i) qRT-PCR analysis of JAG1, HES1, and HEY1 mRNA levels and (j) western blotting analysis of BMSCs after transfected with HDAC1 overexpression under 10% CMS for 3 weeks. (k) ChIP-qPCR analysis of H3 acetylation modification in different JAG1 promoter regions in BMSCs after transfected with HDAC1 overexpression under 10% CMS for 3 weeks. GAPDH was used as an internal control. All results are representative of at least three independent experiments. All the staining data were confirmed by three repeated tests. Data were mean±S.D., *P<0.01. All P-values are based on Student's t-test