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. 2018 Feb 19;8:3257. doi: 10.1038/s41598-018-21601-9

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© The Author(s) 2018

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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Effect of siRNA-mediated depletion of five down-regulated genes (Gli1, Klhl30, Camk2b, Ifitm5, and Hrc) on osteogenic differentiation of MC3T3-E1 cells. (A) The knockdown efficiency of siRNAs targeting five down-regulated genes compared to scramble siRNA was confirmed by RT-PCR in the presence or absence of osteogenic induction at 3 days after transfection. (B) ALP activity after 3 days of osteogenic induction as determined by quantitative ALP activity assay. (C) ALP staining after 4 days of osteogenic induction for MC3T3-E1 cells transfected with 40 pmole siRNAs of Ifitm5, Gli1 and Hrc. (D) Quantification of Alizarin red S staining after 7 days of osteogenic induction. (E,F) Protein expression levels of Runx2, ALP, BMP2/4, OPN, and OPG in as determined by western blot analysis. (G) mRNA expression levels of Runx2, ALP, BMP2, OPN, and OPG as determined by RT-PCR. (H,I) Protein expression levels of β-catenin and GSK-3β as determined by western blot analysis. Specific protein and mRNA expression values were normalized to the expression of β-actin. Results are expressed as mean ± S.D of three independent experiments. (*P < 0.05, **P < 0.01 compared with scramble group).