Journal: Molecular Biology of the Cell

Article Title: Osteopontin inhibits osteoblast responsiveness through the down-regulation of focal adhesion kinase mediated by the induction of low–molecular weight protein tyrosine phosphatase

doi: 10.1091/mbc.E16-10-0716

Figure Lengend Snippet: OPN inhibits HGF- and PDGF-induced cellular events associated with FAK phosphorylation. (A, B) MC3T3-E1 Tet-on OPN cell lines were incubated with or without 2 μg/ml DOXY for 48 h and untreated or treated with either HGF (15 ng/ml) for 20 min or PDGF (10 ng/ml) for 10 min. The prepared cell lysates were separated by SDS–PAGE, and Western blotting was performed with the indicated antibodies. Significant difference from the control by Student’s t test (* p < 0.01). (C, D) MC3T3-E1 cells were treated with 100 ng/ml recombinant OPN protein for 6 h, followed by stimulation and analysis as in A and B. (E) MC3T3-E1 Tet-on OPN cell lines were treated with DOX for 48 h, followed by stimulation with HGF for 2 h. HGF-induced Vdr expression was evaluated by real-time RT-PCR. The same experiments were performed at least three times, producing consistent results. Relative mRNA expression levels in comparison with Rpl13a are shown. Error bars represent SD. Significant difference from the control by Student’s t test (* p < 0.01). (F) MC3T3-E1 cells were pretreated with OPN for 6 h and analyzed as in E. (G) MC3T3-E1 cells were treated with OPN for 3 h, followed by stimulation with 10 ng/ml mouse recombinant PDGF for 30 min. Cells were seeded on fibronectin-coated dishes and fixed with 10% formaldehyde at 60 min. Cells were stained to visualize the cytoplasm by eosin solution. Ten attached cells were randomly selected to measure the cell-spreading area using ImageJ software. Error bars represent SD. Statistical significance was determined by Student’s t test (* p < 0.01). (H, I) MC3T3-E1 cells were cultured in osteogenic differentiation medium for 10 d. The differentiated cells were transfected with OPN siRNA, stimulated by HGF or PDGF, and analyzed as in A and B. (J, K) Differentiated MC3T3-E1 cells were treated with OPN-specific siRNA or anti-OPN antibody, followed by HGF stimulation and analysis as in E and F.

Article Snippet: Recombinant mouse HGF was provided by Mitsubishi Tanabe Pharma (Osaka, Japan).

Techniques: Phospho-proteomics, Incubation, SDS Page, Western Blot, Control, Recombinant, Expressing, Quantitative RT-PCR, Comparison, Staining, Software, Cell Culture, Transfection

Journal: Molecular Biology of the Cell

Article Title: Osteopontin inhibits osteoblast responsiveness through the down-regulation of focal adhesion kinase mediated by the induction of low–molecular weight protein tyrosine phosphatase

doi: 10.1091/mbc.E16-10-0716

Figure Lengend Snippet: LMW-PTP is involved in OPN-induced inactivation of osteoblast reactions. (A–C) MC3T3-E1 Tet-on Flag-LMW-PTP cell lines were incubated with or without 2 μg/ml DOXY for 48 h and either untreated or treated with LIPUS, recombinant HGF (15 ng/ml), or PDGF (10 ng/ml) for the indicated time. The levels of total and phosphorylated FAK were evaluated by Western blotting. Significant difference from the control by Student’s t test (* p < 0.01). (D) MC3T3-E1 Tet-on Flag-LMW-PTP cells were incubated with DOXY for 48 h and stimulated with LIPUS for 20 min. After additional incubation for 2 h, total RNA was isolated and reverse transcribed. Gene expression of Nos1 and Nos2 was analyzed by real-time PCR. The same experiments were performed at least three times with consistent results. Relative mRNA expression levels in comparison with Rpl13a are shown. Error bars represent SD. Significant difference from the control by Student’s t test (* p < 0.01). (E) MC3T3-E1 Tet-on Flag-LMW-PTP cell lines were treated with DOXY for 48 h, followed by stimulation by HGF for 2 h. Real-time PCR analyses were performed as in D. (F–J) MC3T3-E1 cells (F–I) and primary mouse osteoblasts (J) were transiently transfected with either LMW-PTP siRNA or control siRNA. Inhibitory effects of LMW-PTP siRNA on protein expression (F) were examined by Western blotting. After transfection, cells were treated with recombinant OPN for 6 h, followed by stimulation by LIPUS (G, I, J) or HGF (H). Phosphorylation of FAK (G, H) and Nos1 mRNA expression (I, J) was analyzed as in A and D.

Article Snippet: Recombinant mouse HGF was provided by Mitsubishi Tanabe Pharma (Osaka, Japan).

Techniques: Incubation, Recombinant, Western Blot, Control, Isolation, Reverse Transcription, Gene Expression, Real-time Polymerase Chain Reaction, Expressing, Comparison, Transfection, Phospho-proteomics