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. 2018 Apr 11;9:1390. doi: 10.1038/s41467-018-03720-z

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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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Fig. 5 — Increased cell proliferation and delayed osteoblast differentiation and mineralization by MAP2K1 mutations. a MAP2K1 variants found in seven melorheostosis patients do not affect the levels of MAP2K1 transcripts. There was no statistically significant difference in MAP2K1 transcript levels between affected and unaffected osteoblasts (*p < 0.05, paired t-test). Real-time qPCR was carried out in triplicate for each patient sample. b Western blot analysis displays comparable levels of MEK1 protein in affected and unaffected osteoblasts. c Cell proliferation assay using live-cell imaging. Affected and unaffected osteoblasts from melorheostosis patient, Melo-2 (MAP2K1 p.K57N, VAF 45%) were plated at various densities (1000~5000 cells/well) (n = 30). Percent cell confluence is shown at 2-h intervals with symbols indicating mean of replicates (error bars: SEM). Doubling time calculated from the linear phase growth yielded a doubling time of 18 h for affected osteoblasts compared to 54 h for unaffected. Results are representative of two independent experiments. (p < 0.0001). d Western blot analysis shows that affected osteoblasts (lanes 2, 4, and 6) expressed higher level of cyclin D3 compared to unaffected (lanes 1, 3, and 5), correlating with the increased p-ERK1/2 level in affected cells shown in Fig. 4c. Note that levels of cyclin D3 and p-ERK1/2 decreased on day 3 (D3) and day 6 (D6) compared to day 1 (D1), because culture media was not refreshed after day 1, similar to conditions used during the live-cell imaging shown in Fig. 5c. e Alizarin Red S staining of mineralization in osteoblast cultures from patient Melo-18 (MAP2K1 p.K57N, VAF 46%). After 7 weeks of BMP2-stimulated mineralization in vitro, significantly inhibited mineralization was observed in affected cells compared to unaffected. f Real-time qPCR analysis of expression of osteogenic marker genes, RUNX2, COL1A1, and ALPL in osteoblasts from melorheostosis patient Melo-2 (MAP2K1 p.K57N, VAF 45%) after two weeks of osteogenic stimulation. Expression level of RUNX2, COL1A1, and ALPL was significantly lower in affected osteoblasts compared to unaffected. g The RANKL/OPG transcript ratio, an index of osteoclastogenic stimulus, was assessed by real-time qPCR with Melo-2 patient osteoblasts as in Fig. 5f. Significantly higher ratio of RANKL/OPG in affected osteoblasts indicates increased osteoclastogenesis compared to unaffected