Journal: Pflugers Archiv : European journal of physiology

Article Title: Connexin43 enhances Wnt and PGE2-dependent activation of β-catenin in osteoblasts

doi: 10.1007/s00424-019-02295-y

Figure Lengend Snippet: Left, Western blots of active β-catenin, phospho-Akt and connexin43 (Cx43) expression in primary mouse osteoblasts from Gja1flox/flox mice transduced with GFP (control) or Cre-recombinase expressing adenovirus. Right, Western blots of UMR106 osteoblasts transiently transfected with an empty vector control (neo) or a Cx43 overexpression plasmid. Cx43 was examined to validate deletion or overexpression, respectively. GAPDH was used as a loading control. Quantitation of independent experiments is shown below (means ± sd), n=3/group. *, p-value <0.05, **, p-value <0.01.

Article Snippet: UMR106 rat osteosarcoma cells were purchased from ATCC and were cultured as previously described [ 29 ].

Techniques: Western Blot, Expressing, Transduction, Control, Transfection, Plasmid Preparation, Over Expression, Quantitation Assay

Journal: Pflugers Archiv : European journal of physiology

Article Title: Connexin43 enhances Wnt and PGE2-dependent activation of β-catenin in osteoblasts

doi: 10.1007/s00424-019-02295-y

Figure Lengend Snippet: (A)Western blots of active β-catenin in UMR106 cells osteoblasts transiently transfected with an empty vector control (neo) or a Cx43 overexpression plasmid and treated with Wnt3a (50ng/ml, 1 h) or Vehicle (Veh). GAPDH was used as a loading control. Images are from a single exposure of a contiguous membrane. Dotted lines indicate the removal of irrelevant lanes. Quantitation of independent western blotting experiments is shown to the right (means ± sd), n=3/group. *, p < 0.05, **, p < 0.01, ***, p < 0.001. (B) Luciferase reporter assay of β-catenin transcriptional activity from a TCF/Lef reporter (TOPflash) construct following treatment with vehicle (Veh) or Wnt3a (50ng/ml, 4 h). FOPflash contains mutated Tcf/Lef binding sites and represents a negative control. Results are presented as means ± sd, n=3/group. *, p < 0.05, **, p < 0.01, ***, p < 0.001; #, statistical interaction effect of the two variables (Cx43 and Wnt3a) by two-way ANOVA followed by Tukey’s multiple comparison test.

Article Snippet: UMR106 rat osteosarcoma cells were purchased from ATCC and were cultured as previously described [ 29 ].

Techniques: Western Blot, Transfection, Plasmid Preparation, Control, Over Expression, Membrane, Quantitation Assay, Luciferase, Reporter Assay, Activity Assay, Construct, Binding Assay, Negative Control, Comparison

Journal: Pflugers Archiv : European journal of physiology

Article Title: Connexin43 enhances Wnt and PGE2-dependent activation of β-catenin in osteoblasts

doi: 10.1007/s00424-019-02295-y

Figure Lengend Snippet: (A)Western blots of active β-catenin, phospho-Akt, and phospho-GSK3β in UMR106 cells osteoblasts transiently transfected with an empty vector control (neo) or a Cx43 overexpression plasmid and treated with PGE2 (2μM, 1 h) or Vehicle (Veh). GAPDH was used as a loading control. Quantitation of independent western blotting experiments is shown to the right (means ± sd), n=3/group. *, p < 0.05, **, p < 0.01, ***, p < 0.001. (B) Luciferase reporter assay of β-catenin transcriptional activity from a TCF/Lef reporter (TOPflash) construct following treatment with vehicle (Veh) or PGE2 (2μM, 4 h). FOPflash contains mutated TCF/Lef binding sites and represents a negative control. Results are presented as means ± sd, n=3/group. *, p < 0.05, **, p < 0.01, ***, p < 0.001; #, statistical interaction effect of the two variables (Cx43 and PGE2) by two-way ANOVA followed by Tukey’s multiple comparison test.

Article Snippet: UMR106 rat osteosarcoma cells were purchased from ATCC and were cultured as previously described [ 29 ].

Techniques: Western Blot, Transfection, Plasmid Preparation, Control, Over Expression, Quantitation Assay, Luciferase, Reporter Assay, Activity Assay, Construct, Binding Assay, Negative Control, Comparison

Journal: Pflugers Archiv : European journal of physiology

Article Title: Connexin43 enhances Wnt and PGE2-dependent activation of β-catenin in osteoblasts

doi: 10.1007/s00424-019-02295-y

Figure Lengend Snippet: (A)Western blots of active β-catenin, phospho-Akt, and phospho-GSK3β in UMR106 cells osteoblasts transiently transfected with an empty vector control (neo) or a Cx43 overexpression plasmid and treated with PGE2 (2μM, 1 h) or Vehicle (Veh) in the presence or absence of the PI3K inhibitor LY294002 (20μM, 30 min pre-treatment). GAPDH was used as a loading control. Quantitation of independent western blotting experiments is shown to below (means ± sd). *, p < 0.05 relative to the respective DMSO treated control. (B) Luciferase reporter assay of β-catenin transcriptional activity from a TCF/Lef reporter (TOPflash) construct following treatment with vehicle (Veh) or PGE2 (2μM, 4 h) in the presence or absence of the PI3K inhibitor LY294002 (20μM, 30 min pre-treatment). Results are presented as box and whisker plots (min to max), n=20/group. Statistical significance for the three variables (±Cx43, ±PGE2, and ±LY294002) was examined by three-way ANOVA followed by Holm-Sidak multiple comparison test. *, p < 0.05 relative to the respective DMSO treated control. Additional statistical comparisons are in Supplementary Table 1.

Article Snippet: UMR106 rat osteosarcoma cells were purchased from ATCC and were cultured as previously described [ 29 ].

Techniques: Western Blot, Transfection, Plasmid Preparation, Control, Over Expression, Quantitation Assay, Luciferase, Reporter Assay, Activity Assay, Construct, Whisker Assay, Comparison

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Rapid protein kinase A--mediated activation of cyclic AMP-phosphodiesterase by parathyroid hormone in UMR-106 osteoblast-like cells.

doi: 10.1359/jbmr.1997.12.2.172

Figure Lengend Snippet: FIG. 1. (A) Time-dependent effect of bPTH(1–84) on the PDE activity in UMR-106 cells. Control cells (E) and cells treated with 10 nM bPTH(1–84) (F) for indicated times were homogenized and assayed for PDE activity. (B) Con- centration-dependent activation of PDE activity in UMR- 106 cells. The cells were incubated with various concentra- tions of bPTH(1–84) for 20 minutes, homogenized, and assayed for PDE activity. ***p , 0.001, **p , 0.01 as compared with no PTH treatment.

Article Snippet: UMR-106 rat osteosarcoma cells (American Type Culture Collection Rockville, MD, U.S.A.) were grown in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY, U.S.A.) containing 10% fetal bovine serum (FBS; Sigma Chemical Co.) supplemented with 50 IU/ml penicillin and 50 pg/ml streptomycin at 378C in 5% CO2/ 95% air atmosphere on plastic dishes.

Techniques: Activity Assay, Control, Activation Assay, Incubation

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Rapid protein kinase A--mediated activation of cyclic AMP-phosphodiesterase by parathyroid hormone in UMR-106 osteoblast-like cells.

doi: 10.1359/jbmr.1997.12.2.172

Figure Lengend Snippet: FIG. 2. Effect of PKA inhibitor H-8 on bPTH(1–84) and forskolin (FSK)-stimulated PDE activation in UMR-106 cells. The PDE activity stimulated with 10 nM bPTH(1–84) or 5 mM forskolin for 20 minutes was almost completely abolished by H-8 treatment. **p , 0.01 compared with bPTH(1–84) and forskolin treatments with H-8.

Article Snippet: UMR-106 rat osteosarcoma cells (American Type Culture Collection Rockville, MD, U.S.A.) were grown in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY, U.S.A.) containing 10% fetal bovine serum (FBS; Sigma Chemical Co.) supplemented with 50 IU/ml penicillin and 50 pg/ml streptomycin at 378C in 5% CO2/ 95% air atmosphere on plastic dishes.

Techniques: Activation Assay, Activity Assay

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Rapid protein kinase A--mediated activation of cyclic AMP-phosphodiesterase by parathyroid hormone in UMR-106 osteoblast-like cells.

doi: 10.1359/jbmr.1997.12.2.172

Figure Lengend Snippet: FIG. 3. Effect of increasing concentrations of alkaline phosphatase (ALP) on the PDE activity of UMR-106 ho- mogenates. Control cells and cells stimulated for 20 min- utes with 10 nM bPTH(1–84) were homogenized and treated with increasing concentrations of ALP in homoge- nization buffer at 378C for 10 minutes. The ALP-treated homogenates were then assayed for PDE activity. The ef- fect of the ALP treatment was blocked with 50 mM of the ALP inhibitor L-phenylalanine (L-Phe).

Article Snippet: UMR-106 rat osteosarcoma cells (American Type Culture Collection Rockville, MD, U.S.A.) were grown in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY, U.S.A.) containing 10% fetal bovine serum (FBS; Sigma Chemical Co.) supplemented with 50 IU/ml penicillin and 50 pg/ml streptomycin at 378C in 5% CO2/ 95% air atmosphere on plastic dishes.

Techniques: Activity Assay, Control

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Rapid protein kinase A--mediated activation of cyclic AMP-phosphodiesterase by parathyroid hormone in UMR-106 osteoblast-like cells.

doi: 10.1359/jbmr.1997.12.2.172

Figure Lengend Snippet: FIG. 4. Sensitivity of homogenates from bPTH(1–84)- treated UMR-106 cells to specific PDE inhibitors. Cells stimulated with 10 nM bPTH(1–84) for 20 minutes were homogenized and assayed for PDE activity in the presence of increasing concentrations of the cAMP-PDE inhibitor RO 20-1724, cGI-PDE inhibitor milrinone, and cGS-PDE inhibitor MEP-1.

Article Snippet: UMR-106 rat osteosarcoma cells (American Type Culture Collection Rockville, MD, U.S.A.) were grown in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY, U.S.A.) containing 10% fetal bovine serum (FBS; Sigma Chemical Co.) supplemented with 50 IU/ml penicillin and 50 pg/ml streptomycin at 378C in 5% CO2/ 95% air atmosphere on plastic dishes.

Techniques: Activity Assay

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Rapid protein kinase A--mediated activation of cyclic AMP-phosphodiesterase by parathyroid hormone in UMR-106 osteoblast-like cells.

doi: 10.1359/jbmr.1997.12.2.172

Figure Lengend Snippet: FIG. 5. Distribution of PDE activity in soluble and par- ticulate fractions of homogenates from bPTH(1–84)- treated and control cells. UMR-106 cells were treated with 10 nM bPTH(1–84) for 20 minutes and homogenized. Sol- uble enzyme was separated from particulate enzyme by ultracentrifugation at 100,000g for 60 minutes. ***p , 0.001 as compared with control.

Article Snippet: UMR-106 rat osteosarcoma cells (American Type Culture Collection Rockville, MD, U.S.A.) were grown in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY, U.S.A.) containing 10% fetal bovine serum (FBS; Sigma Chemical Co.) supplemented with 50 IU/ml penicillin and 50 pg/ml streptomycin at 378C in 5% CO2/ 95% air atmosphere on plastic dishes.

Techniques: Activity Assay, Control

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Rapid protein kinase A--mediated activation of cyclic AMP-phosphodiesterase by parathyroid hormone in UMR-106 osteoblast-like cells.

doi: 10.1359/jbmr.1997.12.2.172

Figure Lengend Snippet: FIG. 6. Effect of the cAMP-PDE inhibitor RO 20-1724 (RO), cGI-PDE inhibitor milrinone, and PKA inhibitor H-8 on cAMP accumulation in UMR-106 cells. Cells were stimulated with 10 nM bPTH(1–84) (A) or 1 mM forskolin (B) for 10 minutes in the presence of inhibitors and assayed for cellular cAMP. ***p , 0.001, **p , 0.01 as compared with control.

Article Snippet: UMR-106 rat osteosarcoma cells (American Type Culture Collection Rockville, MD, U.S.A.) were grown in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY, U.S.A.) containing 10% fetal bovine serum (FBS; Sigma Chemical Co.) supplemented with 50 IU/ml penicillin and 50 pg/ml streptomycin at 378C in 5% CO2/ 95% air atmosphere on plastic dishes.

Techniques: Control