rat osteoblast differentiation medium rodm  (Cell Applications Inc)

Journal: Nature Communications

Article Title: Somatic activating mutations in MAP2K1 cause melorheostosis

doi: 10.1038/s41467-018-03720-z

Figure Lengend Snippet: MAP2K1 somatic mutations in melorheostotic bone. a Flowchart of genetic analysis. b Schematic of the MAP2K1 exon structure encoding MEK1 protein domains. The three mutations from the eight patients are clustered in the negative regulatory domain. c Quantification of mutant allele abundance by ddPCR in unaffected and affected bone biopsies (top row), as well as in cultured osteoblasts from the respective biopsies (bottom row), from patient Melo-2 identified by WES to have a p.K57N mutation in affected bone. Each dot represents a droplet, with blue being mutant positive, green being wildtype positive, and orange being positive for both. Color-matched numbers correspond to count of droplets per quadrant. The boxed number is the fractional abundance of mutant allele in each sample

Article Snippet: Following confluence, osteoblasts were grown in osteoblast differentiation medium (50 μg/mL L -ascorbic acid, 10 nM dexamethasone, and 2.5 mM β-glycerophosphate), with or without 100 ng/mL recombinant BMP2 (#355-BM, R&D Systems, Minneapolis, MN), refreshing osteogenic media every 3 days for 7 weeks.

Techniques: Mutagenesis, Cell Culture

Journal: Nature Communications

Article Title: Somatic activating mutations in MAP2K1 cause melorheostosis

doi: 10.1038/s41467-018-03720-z

Figure Lengend Snippet: Activation of the MEK1-ERK1/2 pathway by MAP2K1 mutations. a Immunohistochemical analysis of ERK1/2 activation in bone tissues from a melorheostosis patient. Left column shows sections of colon cancer stained with p-ERK1/2-specific antibodies. Marked brown staining is visible. Middle column shows a section of normal bone stained in a similar fashion. Right column shows representative section from melorheostotic bone (Melo-18, MAP2K1 p.K57N, VAF 46%) stained with p-ERK1/2-specific antibodies. Osteocytes stain brown surrounded by woven bone. Cells positive for p-ERK1/2 are also seen in the periosteum. Inset shows high-power view of positively staining cells. A multinucleated osteoclast which does not stain for p-ERK1/2 is marked by the solid arrow. Cells negative for p-ERK1/2 are noted by the open arrows. See Supplementary Figure for staining of SW48 colon cancer cells, which harbor the MAP2K1 p.K56P mutation. b p-ERK1/2-specific flow cytometry analysis. Affected and unaffected osteoblasts from Melo-2 ( MAP2K1 p.K57N, VAF 45%) were stimulated with serum with or without MEK inhibitor U0126. Two peaks in the histogram indicates cell subpopulations of distinct level of p-ERK1/2 upon serum-stimulation in osteoblasts from affected bone (red), cells from unaffected bone (blue) only showed a single peak. U0126 reduced p-ERK1/2 in cells from affected bone. Cells stained with matching rabbit IgG isotype control are also shown (gray). The geometrical mean channel fluorescence is shown below for the high and low peaks of p-ERK1/2 marked on the histograms. c Western blot analysis of osteoblasts from affected and unaffected bone of patient Melo-2 ( MAP2K1 p.K57N, VAF 45%) shows increased ERK1/2 activation (p-ERK1/2) by MEK1 mutation in affected osteoblasts (lane 7 of p-ERK1/2 blot), as compared to unaffected osteoblasts (lane 3), by serum-stimulation. Inhibition of MEK1 with U0126 significantly diminished p-ERK1/2 in both affected and unaffected, but the level of p-ERK1/2 was still higher in affected osteoblasts compared to unaffected (lanes 4 and 8). Quantification data of band intensities are shown in a table. COX IV was used as control for equal amount protein loading

Article Snippet: Following confluence, osteoblasts were grown in osteoblast differentiation medium (50 μg/mL L -ascorbic acid, 10 nM dexamethasone, and 2.5 mM β-glycerophosphate), with or without 100 ng/mL recombinant BMP2 (#355-BM, R&D Systems, Minneapolis, MN), refreshing osteogenic media every 3 days for 7 weeks.

Techniques: Activation Assay, Immunohistochemical staining, Staining, Mutagenesis, Flow Cytometry, Control, Fluorescence, Western Blot, Inhibition

Journal: Nature Communications

Article Title: Somatic activating mutations in MAP2K1 cause melorheostosis

doi: 10.1038/s41467-018-03720-z

Figure Lengend Snippet: 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. . 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

Article Snippet: Following confluence, osteoblasts were grown in osteoblast differentiation medium (50 μg/mL L -ascorbic acid, 10 nM dexamethasone, and 2.5 mM β-glycerophosphate), with or without 100 ng/mL recombinant BMP2 (#355-BM, R&D Systems, Minneapolis, MN), refreshing osteogenic media every 3 days for 7 weeks.

Techniques: Western Blot, Proliferation Assay, Live Cell Imaging, Staining, In Vitro, Expressing, Marker