murine osteoblastic progenitor cell line mc3t3-e1  (BioResource International Inc)

Journal: The Journal of Biological Chemistry

Article Title: The secreted protein DEL-1 activates a β3 integrin–FAK–ERK1/2–RUNX2 pathway and promotes osteogenic differentiation and bone regeneration

doi: 10.1074/jbc.RA120.013024

Figure Lengend Snippet: DEL-1 promotes osteogenic differentiation and mineralization in a manner dependent on FAK and ERK1/2. A, MC3T3-E1 osteoblastic progenitor cells were incubated in growth medium with 1 μg/ml DEL-1-Fc or DEL-1[RGE]-Fc or equal molar concentration of Fc control for the indicated times. Immunoblot analysis was performed with specific antibodies against phosphorylated and total FAK, AKT, and ERK1/2 as well as against Runx2 and β-actin (loading control). B and C, MC3T3-E1 cells were cultured in osteogenic medium in the presence or DEL-1-Fc (1 μg/ml) or equal molar amounts of Fc control, DEL-1[RGE]-Fc, or DEL-1[E1–3]-Fc. In some DEL-1-Fc–treated groups, the cells were pretreated with PF-562271 (1 μm) or U0126 (10 μm); these inhibitors were added 1 h prior to DEL-1-Fc. Medium was changed every 3 days and was supplemented, as appropriate, with fresh DEL-1-Fc (or mutants/controls thereof) in the presence or absence of fresh signaling inhibitors. Shown are representative images of mineralized nodule formation, detected by Alizarin Red S staining, at day 15 of differentiation (B, left) and the mineralization area in each culture quantified and expressed as a percentage of the total area (B, right). C, analysis of MC3T3-E1 cells (treated as in B) for the expression of Runx2 (at day 6), Sp7 (at day 9), and Bglap (at day 12), typical early, middle, and late osteogenic markers, respectively, using qPCR. Data were normalized to Gapdh mRNA and expressed relative to medium-only–treated control, set as 1. D, Western blot analysis of Runx2 protein expression at 48 h in MC3T3-E1 cells, incubated in growth medium treated with DEL-1-Fc, in the presence or absence of the indicated concentrations of U0126 or PF-562271, which were added 1 h earlier than DEL-1-Fc. Numerical data are means ± S.D. (error bars) (n = 6 cultures/group). **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; NS, nonsignificant (one-way ANOVA and Tuckey's post-test).

Article Snippet: The murine osteoblastic progenitor cell line (MC3T3-E1 subclone 4) was purchased from ATCC (Manassas, VA) (catalog no. CRL-2593).

Techniques: Incubation, Concentration Assay, Control, Western Blot, Cell Culture, Staining, Expressing

Journal: The Journal of Biological Chemistry

Article Title: The secreted protein DEL-1 activates a β3 integrin–FAK–ERK1/2–RUNX2 pathway and promotes osteogenic differentiation and bone regeneration

doi: 10.1074/jbc.RA120.013024

Figure Lengend Snippet: Endogenous DEL-1 induces osteogenic differentiation in primary calvarial osteoblast progenitors through its RGD motif. Primary osteoblastic progenitor cells were isolated from the calvariae of 3-day-old WT, Del1KO, or Del1RGE/RGE mice. The cells were cultured in osteogenic medium in the presence (or not) of DEL-1-Fc (1 μg/ml) or equal molar amounts of Fc control, DEL-1[RGE]-Fc, or DEL-1[E1–3]-Fc and were compared for mineralized nodule formation (A and B) and osteogenic gene expression (C). Shown are representative images of mineralized nodule formation, detected by Alizarin Red S staining, on day 15 of differentiation (A) and mineralization area in each culture quantified and expressed as a percentage of the total area (B). C, primary calvarial osteoblast progenitors from the same strains of mice, treated similarly as above, were assayed by qPCR for expression of Runx2 (at day 6), Sp7 (at day 9), and Bglap (at day 12), typical early, middle, and late osteogenic markers, respectively. Data were normalized to Gapdh mRNA and expressed relative to the medium-only–treated groups of the WT cells, set as 1. D–G, primary osteoblastic progenitor cells, isolated from the calvariae of 3-day-old WT mice, were cultured in osteogenic medium in the presence (or not) of DEL-1-Fc (1 μg/ml) with or without cilengitide (5, 10, 20, 30, or 40 μm) or 40 μm RGD control peptide and assayed for mineralization nodule formation (D and E) and osteogenic gene expression (F). Shown are representative images of mineralized nodule formation, detected by Alizarin Red S staining, on day 15 of differentiation (D), and the mineralization area in each culture was quantified and expressed as a percentage of the total area (E). F, primary calvarial osteoblast progenitors from WT mice were treated as above and assayed by qPCR for expression of Runx2 (at day 6), Sp7 (at day 9), and Bglap (at day 12), respectively. Data were normalized to Gapdh mRNA and expressed relative to the control peptide-only–treated group, set as 1. G, Western blot analysis of Runx2 protein expression at 48 h in WT primary osteoblastic progenitor cells incubated in growth medium in the presence (or not) of DEL-1-Fc (1 μg/ml) with cilengitide (5, 10, 20, 30, and 40 μm) or control peptide (40 μm), which were added 1 h earlier than DEL-1-Fc. β-Actin served as loading control. Data are means ± S.D. (error bars) (n = 6 cultures/group). **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; NS, nonsignificant (one-way ANOVA and Tukey's post-test).

Article Snippet: The murine osteoblastic progenitor cell line (MC3T3-E1 subclone 4) was purchased from ATCC (Manassas, VA) (catalog no. CRL-2593).

Techniques: Isolation, Cell Culture, Control, Gene Expression, Staining, Expressing, Western Blot, Incubation