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. 2019 Jun 26;9:9307. doi: 10.1038/s41598-019-45793-w

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© The Author(s) 2019

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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Increased ALP activity, calcium deposition and its markers gene by FN. Osteoblast differentiation was induced with osteogenic induction medium (HSCG) in the presence of different concentrations (1–15 µM) of FN and incubated for different days. (a) Cells were fixed in formalin and stained with Alizarin Red S for 30 min. Cell images were obtained by EVOS cell image system at 10X. (b) Cell extracts were prepared and used for ALP activity at 24, 48, and 72 h. (c) Alizarin Red S stain was extracted and quantified on day 2, 4 and 6 according to the kit protocol. (d) The total RNA was extracted and reverse transcribed for quantification of mRNA by qPCR. ALP, RUNX2 and osteocalcin mRNA levels were determined after normalization with β-actin. All values represent mean ± SEM, n = 6 for ALP, calcium level and qPCR. Different letters a, b, c and d within a column indicate significant differences between treatment and non-treatment (p < 0.05). Statistical significance was performed using the general linear model with multivariate, post hoc test and comparisons with respective controls.