Journal: Journal of cellular biochemistry

Article Title: Expression of the ectodomain-releasing protease ADAM17 is directly regulated by the osteosarcoma and bone-related transcription factor RUNX2

doi: 10.1002/jcb.26832

Figure Lengend Snippet: To identify genes that immediately respond to osteogenic stimuli, we retrieved microarray data were retrieved for experiments with mouse C2C12 mesenchymal cells that were treated with 300 ng/ml of BMP-2 and analysed at distinct time points (0, 4, 8, 12, 16, 20, and 24 h). Data on ADAM genes were filtered for genes that show more than a 2 fold change in gene expresseion. This analysis revealed that the Adam17, Adam10 and Adam9 genes are upregulated for more than 2-fold (A), while three others are downregulated (Adam8, Adam15, Adam19) (B). (C) The bar graph shows average expression values (in RPKM; STD as error bar for n=3 human donors) that were rank ordered for relative expression based on RNA-seq analysis of human osteoblastic bone-derived cells from three different donors. The pie chart presented in the inset shows that the six most highly expressed ADAM genes (including the BMP-responsive ADAMs ADAM17, ADAM10 and ADAM9, which are presented in color) account for almost all (~97%) ADAM-related transcripts. (D) Results of RNA-seq analysis for each individual donor and select osteosarcoma cell lines (SaOS-2, MG63 and U2OS) as indicated. (E) Visual presentation and validation of gene expression data using semi-quantitative RT-PCR and ethidium bromide staining. ADAM17, ADAM10 and ADAM9 gene expression was assessed as indicated in human SAOS-2, MG63, U2OS, HOS, G292 and 143B immature osteoblast cells. The data shown are representative of three experiments with similar outcomes. The graphs show quantification of the RT-PCR data relative to Gapdh mRNA (D). All data are presented as mean ± SEM from three independent experiments.

Article Snippet: Mouse MC3T3–E1 osteoblasts, mouse pre-myogenic mesenchymal C2C12 precursor cells, rat osteosarcoma ROS17/2.8 cells and human osteosarcoma cell lines (SAOS-2, MG63, U2OS, G292, HOS and 143B cells) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Microarray, Expressing, RNA Sequencing, Derivative Assay, Biomarker Discovery, Gene Expression, Quantitative RT-PCR, Staining, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of cellular biochemistry

Article Title: Expression of the ectodomain-releasing protease ADAM17 is directly regulated by the osteosarcoma and bone-related transcription factor RUNX2

doi: 10.1002/jcb.26832

Figure Lengend Snippet: Adam17 and Runx2 expression was assessed in mouse Runx2-null osteoprogenitor cells (Runx2−/−) and mouse MC3T3–E1 pre-osteoblasts cells, as well as rat ROS17/2.8 and human SAOS-2 osteosarcoma cells. Protein and mRNA levels were evaluated by western blot analysis (A, and down graph B) and RT-PCR (C, and down graph D), respectively. Runx2-null cells were infected with an adenovirus vector expressing RUNX2 or GFP (control) as indicated (E and G). Alternativelly, cells were transiently transfected with different concentrations (0.5, 1, 2.5, 5, and 10 μg of DNA) of pcDNA-Runx2 or pcDNA-empty vector (control) (I). Runx2-null cells expressing Adam17 and Runx2 mRNA (E, I, and down graph F and J) and protein levels (G, and down graph H) were evaluated by RT-PCR and western blot analysis, respectively. The data shown are representative of three experiments with similar outcomes. Adam17 and Runx2 mRNA and protein values were normalized to Gapdh and Actin, respectively. All data are presented as mean ± SEM from three independent experiments. *P<0.05 and **P<0.01.

Article Snippet: Mouse MC3T3–E1 osteoblasts, mouse pre-myogenic mesenchymal C2C12 precursor cells, rat osteosarcoma ROS17/2.8 cells and human osteosarcoma cell lines (SAOS-2, MG63, U2OS, G292, HOS and 143B cells) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Infection, Plasmid Preparation, Control, Transfection

Journal: Journal of cellular physiology

Article Title: Mitotic inheritance of mRNA facilitates translational activation of the osteogenic-lineage commitment factor Runx2 in progeny of osteoblastic cells

doi: 10.1002/jcp.25188

Figure Lengend Snippet: (A) Osteoblastic cell (hFOB) and osteosarcoma cells (U2OS, G292 and HOS) were subjected to RISH analysis. Scale bars: 20 µm, 100X oil objective, numerical aperture = 1.25. (B) Runx2 RISH signal intensity was determinede by quantitative image analysis in progeny cells at the last step of cytokinesis (n=10). Runx2 mRNA (RISH, left image) exhibits a partitioning coefficient equivalent to that of DNA (mitotic chromosomes staining, right image), demonstrating that this transcript segregates symmetrically in progeny cells upon cell division. Student’s t test was performed to assess the significance of the observed differences.

Article Snippet: Cell culture Mouse pre-osteoblastic MC3T3-E1 cells, human osteoblastic hFOB cells and human osteosarcoma cells (U2OS, G292, and HOS) were maintained as indicated in αMEM or DMEM culture medium (Gigco, Life Technologies, Grand Island, NY, USA) supplemented with 10–15% fetal bovine serum (FBS) plus 2 mM L-glutamine and a penicillin-streptomycin cocktail at 37°C and 5% CO 2 according to ATCC recommendations.

Techniques: Staining