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

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 Virology

Article Title: Computational and Functional Analysis of the Virus-Receptor Interface Reveals Host Range Trade-Offs in New World Arenaviruses

doi: 10.1128/JVI.01408-15

Figure Lengend Snippet: An extended region of TfR1 determines species specificity of Machupo virus entry. (A) An alignment of one portion of the TfR1 apical domain is shown for host and nonhost species. The amino acid numbering corresponds to human TfR1. The three residues experiencing recurrent positive selection, as previously defined (28), are highlighted in yellow. In blue type, a strategy is summarized that was previously used to map the Machupo virus-binding interface on TfR1 (19). In that previous work, this region was swapped from the human TfR1 (RLVYL) into the house mouse (M. musculus) TfR1, replacing the corresponding 4 residues (NLDP). In the present work, residues in the C. callosus TfR1 were swapped into the rat TfR1 to create the rat-short (red type) and rat-long (red plus green type) chimeric TfR1s shown in the bottom two lines. Also shown is the position of the L212V human SNP discussed in this study. (B) The cocrystal structure shows the interaction between Machupo virus GP1 (blue) and the human TfR1 apical domain (gray) (PDB code 3KAS) (18). The residue positions mutated in the rat-short TfR1 chimera are shown in brown. The residue positions mutated in the rat-long chimera are shown in green and brown. (C) Canine D17 cells were transduced to stably express the wild-type and chimeric TfR1s with C-terminal FLAG tags. Cells were infected with increasing volumes of Machupo pseudovirus (GFP-encoding retrovirus pseudotyped with Machupo virus glycoprotein [GP]). Cells were monitored for GFP expression to determine the percentage of infected cells. Error bars indicate standard deviations from three technical replicates. The experiment was performed 3 times with similar results seen in all experiments; the graph represents data from one experiment. A t test was performed to determine if differences between mean values were statistically significant (*, <1e−8). Cell surface expression of TfR1 was monitored with a FLAG antibody via flow cytometry (inset) concurrently with measurement of GFP signal. Expression is given as mean fluorescence intensity (MFI). C.c, C. callosus; r-l, rat-long; r-s, rat-short; r, rat. (D) A Coomassie blue-stained SDS-PAGE gel shows purified Machupo virus GP1 fused to the human IgG1 Fc fragment. (E) Coomassie blue-stained SDS-PAGE gels show the five purified TfR1 proteins. (F) ELISA comparing the relative binding affinities of Machupo virus GP1 to each of the purified TfR1s. Purified TfR1 was bound to wells, and then GP1 was incubated at decreasing concentrations to determine the relative binding affinities. Error bars indicate standard deviations from three technical replicates. The experiment was performed 2 times with similar results seen in the two experiments; the graph represents data from one experiment. Curves were fitted using 4-parameter nonlinear regression.

Article Snippet: Human embryonic kidney 293T cells (ATCC) and canine osteosarcoma D17 cells (ATCC) were maintained in Dulbecco's modified Eagle's medium (Cellgro) supplemented with 10% fetal bovine serum (Gibco), 100 units ml −1 penicillin, 100 μg ml −1 streptomycin, and 2 mM l -glutamine (Cellgro).

Techniques: Virus, Selection, Binding Assay, Residue, Stable Transfection, Infection, Expressing, Flow Cytometry, Fluorescence, Staining, SDS Page, Purification, Enzyme-linked Immunosorbent Assay, Incubation

Journal: Journal of Virology

Article Title: Computational and Functional Analysis of the Virus-Receptor Interface Reveals Host Range Trade-Offs in New World Arenaviruses

doi: 10.1128/JVI.01408-15

Figure Lengend Snippet: Virus-specific effects on entry through TfR1. (A) The Chapare virus GP1 (blue) was homology modeled on the Machupo virus GP1 crystal structure (magenta; PDB code 3KAS). The Machupo virus GP1 loop 10 and surrounding residues are shown in light pink where divergence in structure is seen in the Chapare virus GP1. Similar results were seen with Sabia and Junin virus models (data not shown). (B) An alignment illustrates the C-terminal region of GP1 from Machupo, Junin, Sabia, Chapare, and Guanarito arenaviruses. Residues identical to those in Machupo virus have been replaced with dots. Machupo virus GP1 sites that make contact with TfR1 (18) are indicated with red asterisks. They fall in loop 7, loop 10, and two upstream regions which are not shown. Loop 10 is longer in Machupo virus than in all other viruses. The numbering scheme is based on the Machupo virus protein. Note that the alignment differs somewhat from a similar alignment shown in reference 18. The differences in these alignments concern the exact placement of insertions and deletions but are within the range to be expected from different alignment approaches. This alignment was generated using MAFFT (47, 48). (C and D) Canine D17 cells were transduced to stably express the wild-type and chimeric TfR1s with C-terminal FLAG tags. Cells were infected with increasing volumes of GFP-encoding retrovirus pseudotyped with Sabia (C) or Chapare (D) virus glycoprotein (GP). Cells were monitored for GFP expression to determine the percentage of infected cells. Error bars indicate standard deviations from three technical replicates. Experiments were performed 2 times, with similar results seen in the two experiments; the graph represents data from one experiment. A t test was performed to determine if differences between mean values were statistically significant (*, <1e−5; **, <1e−10). Cell surface expression of TfR1 was monitored with a FLAG antibody via flow cytometry (inset) concurrently with measurement of GFP signal. Expression is given as mean fluorescence intensity (MFI). See legend to Fig. 2C for definitions of abbreviations.

Article Snippet: Human embryonic kidney 293T cells (ATCC) and canine osteosarcoma D17 cells (ATCC) were maintained in Dulbecco's modified Eagle's medium (Cellgro) supplemented with 10% fetal bovine serum (Gibco), 100 units ml −1 penicillin, 100 μg ml −1 streptomycin, and 2 mM l -glutamine (Cellgro).

Techniques: Virus, Generated, Stable Transfection, Infection, Expressing, Flow Cytometry, Fluorescence

Journal: Journal of Virology

Article Title: Computational and Functional Analysis of the Virus-Receptor Interface Reveals Host Range Trade-Offs in New World Arenaviruses

doi: 10.1128/JVI.01408-15

Figure Lengend Snippet: Human TfR1 L212V SNP has an opposite effect on the entry of related arenaviruses. (A) The L212 residue in TfR1 contacts loop 10 in the Machupo virus GP1 (PDB code 3KAS) (18). (B) Canine D17 cells were transduced to stably express the human TfR1 either with L212 or bearing the human SNP mutation, L212V, both with a C-terminal FLAG tag. Cells were infected with increasing volumes of a GFP-encoding retrovirus pseudotyped with Machupo virus glycoprotein. Cells were monitored for GFP expression to determine the percentage of infected cells. Error bars indicate standard deviations from three technical replicates. The experiment was performed 2 times with similar results seen in the two experiments; the graph represents data from one experiment. A t test was performed to determine if differences between mean values were statistically significant (*, <1e−3). Cell surface expression of TfR1 was monitored with a FLAG antibody via flow cytometry (right) concurrently with measurement of GFP signal. Expression is given as mean fluorescence intensity (MFI). (C) ELISA comparing the relative binding affinities of the purified human L212 and L212V TfR1s to Machupo virus GP1. Purified TfR1 was bound to wells. GP1 was then incubated at decreasing concentrations to determine the relative binding affinities. Error bars indicate standard deviations from three technical replicates. The experiment was performed 2 times with similar results seen in the two experiments; the graph represents data from one experiment. Curves were fitted using 4-parameter nonlinear regression. pbs, phosphate-buffered saline. (D) ELISA comparing the relative binding affinities of the purified human L212 and L212V TfR1 to iron-loaded human transferrin. For the ELISA, purified TfR1 was bound to wells. Iron-loaded TfR1 was incubated at decreasing concentrations to determine the relative binding affinities. Error bars indicate standard deviations from three technical replicates. The experiment was performed 2 times with similar results seen in the two experiments; the graph represents data from one experiment. Curves were fitted using 4-parameter nonlinear regression. (E and F) The experiment is the same as that in panel B. In this case, cells were infected over increasing volumes with a GFP-encoding retrovirus pseudotyped with Junin (E) or Sabia (F) virus glycoprotein.

Article Snippet: Human embryonic kidney 293T cells (ATCC) and canine osteosarcoma D17 cells (ATCC) were maintained in Dulbecco's modified Eagle's medium (Cellgro) supplemented with 10% fetal bovine serum (Gibco), 100 units ml −1 penicillin, 100 μg ml −1 streptomycin, and 2 mM l -glutamine (Cellgro).

Techniques: Residue, Virus, Stable Transfection, Mutagenesis, FLAG-tag, Infection, Expressing, Flow Cytometry, Fluorescence, Enzyme-linked Immunosorbent Assay, Binding Assay, Purification, Incubation, Saline