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. 2012 Oct 25;8(10):1310–1322. doi: 10.7150/ijbs.5136

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© Ivyspring International Publisher. This is an open-access article distributed under the terms of the Creative Commons License (http://creativecommons.org/licenses/by-nc-nd/3.0/). Reproduction is permitted for personal, noncommercial use, provided that the article is in whole, unmodified, and properly cited.

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Fig 3 — Overexpression of Col24α1 significantly increases osteoblast differentiation and mineralization. MC3T3-E1 cells were infected with Control or Col-O retroviruses for 24 hrs and then induced with OS media for 7 and 14 days. (A) Western blot analysis. The expression level of Col24α1 in the Col-O group is increased 5.4 fold (7 days) and 4.1 fold (14 days) than that in the control. (B) Quantitative real-time RT-PCR analysis of osteoblast marker genes Runx2, BSP, ALP, and OCN in MC3T3-E1 cells induced with OS media for 7 days. For all genes the mRNA levels were significantly increased in Col-O group as compared with the Control. N=6, p < 0.05; Control vs Col-O for all genes. (C) Alizarin red staining. There is an increase in calcium deposits for the overexpression group as compared to the control. (D) The quantitative analysis of Alizarin red staining as seen in C. N=6, p<0.05; a: Control vs Col-O. (E) Von Kossa staining method shows increased phosphate ion deposits in mineralized matrix. (F) ALP activity. Following infection of MC3T3-E1 cells with Col-O or Control retroviruses they were induced with OS media for 7 days. The Col-O group shows an increase in activity as compared to the Control group. N=6, p<0.05; a: Control vs Col-O. Unless indicated otherwise normalization was performed against GAPDH levels for the control group in the same reaction.