Journal: Journal of Biological Chemistry

Article Title: Bone-related Genes Expressed in Advanced Malignancies Induce Invasion and Metastasis in a Genetically Defined Human Cancer Model

doi: 10.1074/jbc.m211498200

Figure Lengend Snippet: FIG. 2. Phenotype associated with expression with osteonec- tin and osteoactivin. Genetically defined glioma cells were engi- neered to express vector control (VEC), osteonectin (ON), or osteoactivin (OA). As shown in A, cell cycle analysis by flow cytometry revealed that the expression of osteonectin and osteoactivin was associated with minimal changes in the cell cycle fractions. As shown in B, DNA syn- thesis measured by thymidine incorporation revealed minimal impact by osteonectin or osteoactivin expression. As shown in C, equal amounts of protein from genetically defined human glioma cells expressing os- teonectin or osteoactivin were subjected to Western analysis. Osteonec- tin expression was not associated with changes relative to vector con- trol, whereas osteoactivin expression was associated with a moderate increase in EGFR expression and decrease of PDGFR- expression. As shown in D, transformation of human astrocytes expressing SV40 T antigen and the human telomerase catalytic subunit with vector con- trol, osteonectin, osteoactivin, or oncogenic Ha-ras (RAS) was tested in a soft agar colony formation assay.

Article Snippet: Primary antibodies for anti-osteonectin (Hematologic Technologies, Essex Junction, VT), anti-GPNMB (gift of Carol Wikstrand, Duke University), antiactin (Santa Cruz Biotechnology, Santa Cruz, CA), anti-tubulin (Sigma), anti-MMP-9 (Calbiochem), anti-MMP-3 (Calbiochem), anti-MMP-2 (Calbiochem), anti-epidermal growth factor receptor (EGFR) (gift of Carol Wikstrand, Duke University), anti-platelet-derived growth factor receptor (PDGFR ) (Santa Cruz Biotechnology), anti-PDGFR (Santa Cruz Biotechnology), or anti-insulin-like growth factor-1 receptor (IGF-1R) (Calbiochem) antibodies were used.

Techniques: Expressing, Plasmid Preparation, Control, Cell Cycle Assay, Flow Cytometry, Western Blot, Transformation Assay, Soft Agar Assay

Journal: eLife

Article Title: Integrin alpha11 is an Osteolectin receptor and is required for the maintenance of adult skeletal bone mass

doi: 10.7554/elife.42274

Figure Lengend Snippet: Figure 3. Osteogenic differentiation in response to Osteolectin requires b-catenin. (A and B) MC3T3-E1 cells, hBMSC#1 cells, and hBMSC#2 cells were transferred into osteogenic differentiation medium with PBS or 30 ng/ml recombinant mouse Osteolectin, as well as DMSO or 200 nM of the GSK3 inhibitor AZD2858. (A) Cells were lysed 24 hr later and immunoblotted for phospho-GSK3, b-catenin, and Actin. (B) Alizarin red staining after 14 days (MC3T3-E1 cells) or 21 days (hBMSC cells) to quantify osteoblast differentiation and mineralization (n = 3 independent experiments). (C and D) MC3T3- E1 cells, hBMSC#1 cells, and hBMSC#2 cells were transferred into osteogenic differentiation medium with PBS or 30 ng/ml Osteolectin, as well as DMSO or 200 nM of the b-catenin inhibitor IWR-1-endo. (C) Cells were lysed 24 hr later and immunoblotted for phospho-GSK3, b-catenin, and Actin. (D) Alizarin red staining after 14 days (MC3T3-E1 cells) or 21 days (hBMSC cells) to quantify osteoblast differentiation and mineralization (n = 3 independent experiments). All numerical data reflect mean ±standard deviation. The statistical significance of differences was determined with two-way ANOVAs with Tukey’s multiple comparisons tests. DOI: https://doi.org/10.7554/eLife.42274.007 The following source data is available for figure 3:

Article Snippet: DOI: https://doi.org/10.7554/eLife.42274 18 of 31 Continued Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody goat polyclonal anti-Mouse Leptin R, Biotin R and D Systems AF497 (1:200) Antibody mouse monoclonal anti-Streptavidin PE Biolegend 410504 (1:500) Peptide, recombinant protein Bovine serum albumin Sigma-Aldrich A3156 Peptide, recombinant protein recombinant human pro-Collagen I a1 R and D Systems 6220 CL Peptide, recombinant protein recombinant human osteolectin PMID: 27976999 Peptide, recombinant protein recombinant mouse osteolectin PMID: 27976999 Peptide, recombinant protein recombiant Integrin a11b1 protein R and D Systems 6357-AB Peptide, recombinant protein recombiant Integrin a10b1 protein R and D Systems 5895-AB Peptide, recombinant protein recombiant Integrin aVb3 protein R and D Systems 3050-AV Peptide, recombinant protein recombiant Integrin aVb1 Protein R and D Systems 6579-AV Peptide, recombinant protein recombiant Integrin a4b1 protein R and D Systems 5668-A4 Peptide, recombinant protein recombiant Integrin a9b1 protein R and D Systems 5438-A9 Peptide, recombinant protein recombiant Integrin aIIbb3 Protein R and D Systems 7148-A2 Peptide, recombinant protein recombiant Integrin aMb2 Protein R and D Systems 4047-AM Chemical compound, drug DAPI Life Technologies D1306 Chemical compound, drug TRIzol LS Reagent Invitrogen 10296028 Chemical cmpound, drug Collagenase, Type 1 Worthington LS004196 Chemical compound, drug Dispase II Roche Diagnostic D4693 Chemical compound, drug DNase I Sigma-Aldrich 10 104 159 001 Chemical compound, drug IWR-1-endo Sigma-Aldrich 681669 Chemical compound, drug AZD2858 Selleck S7253 Chemical compound, drug Y-27632 Rock inhibitor Selleck S1049 Chemical compound, drug 4% paraformaldehyde in PBS Thermo Fisher Scientific J19943-K2 Continued on next page Shen et al. eLife 2019;8:e42274.

Techniques: Recombinant, Staining, Standard Deviation