Journal:

Article Title: Glutamate Suppresses Osteoclastogenesis through the Cystine/Glutamate Antiporter

doi: 10.2353/ajpath.2007.061039

Figure Lengend Snippet: Expression of cystine/Glu antiporter and regulation by RANKL in osteoclasts. A: mRNA was isolated from preosteoclasts and mature osteoclasts for subsequent RT-PCR using primers specific for xCT or 4F2hc subunit. Typical pictures are shown in the figure with similar results in three separate determinations. Br, whole brain. B: Preosteoclasts cultured for an additional 6 hours in the presence of M-CSF alone were incubated with 1 μmol/L [14C]cystine at 2°C or 37°C for 10 minutes. C: Preosteoclasts were cultured in either the presence or absence of 20 ng/ml RANKL for 24 hours, followed by the isolation of mRNA and subsequent RT-PCR using primers specific for xCT or 4F2hc subunit. D: Preosteoclasts were cultured in either the presence or absence of 20 ng/ml RANKL for 24 hours, followed by the homogenization and subsequent centrifugation at 100,000 × g for immunoblotting analysis using an antibody against xCT subunit. Typical pictures are shown in the figure, whereas similar results were invariably obtained in at least three independent determinations. E: Preosteoclastic RAW264.7 cells were transiently transfected with xCT promoter (a) or xCT promoter containing 3′ UTR (b) of xCT mRNA, followed by cultivation in either the presence or absence of 20 ng/ml RANKL for 48 hours and subsequent cell harvest for determination of the luciferase activity. Values are the mean ± SE from four different experiments. **P < 0.01, significantly different from control value obtained in the absence of RANKL.

Article Snippet: TA cloning vector, pGL3 basic vector, and Dual luciferase assay system were purchased from Promega (Madison, WI).

Techniques: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Incubation, Homogenization, Centrifugation, Western Blot, Transfection, Luciferase, Activity Assay, Control

Journal:

Article Title: Glutamate Suppresses Osteoclastogenesis through the Cystine/Glutamate Antiporter

doi: 10.2353/ajpath.2007.061039

Figure Lengend Snippet: Effect of Glu on cell differentiation of preosteoclastic RAW264.7 cells. A: RAW264.7 cells were cultured for 4 days with 20 ng/ml RANKL in either the presence or absence of Glu at a concentration range from 0.1 to 1 mmol/L, followed by determination of the number of TRAP-positive MNCs. Typical pictures are shown in the left panel, whereas quantitative data are shown in the right panel. **P < 0.01, significantly different from control value obtained in the absence of Glu. B: RAW264.7 cells were cultured with RANKL in either the presence or absence of different test drugs, followed by determination of the number of TRAP-positive MNCs. **P < 0.01, significantly different from the value obtained in the absence of Glu. ##P < 0.01, significantly different from the value obtained in the presence of Glu alone. C: RAW264.7 cells were transiently transfected with the reporter plasmid of NF-κB, followed by further cultivation with RANKL at 1 to 50 ng/ml in either the presence or absence of Glu at 0.1 to 1 mmol/L for 48 hours and subsequent cell harvest for determination of the luciferase activity. **P < 0.01, significantly different from control value obtained in the absence of both RANKL and Glu. ##P < 0.01, significantly different from the value obtained in the presence of 10 ng/ml RANKL alone. D: Cells were transiently transfected with the reporter plasmid of NF-κB, followed by further cultivation with 10 ng/ml RANKL and 500 μmol/L Glu in either the presence or absence of homocysteic acid or GSH at 500 μmol/L for 48 hours and subsequent cell harvest for determination of the luciferase activity. **P < 0.01, significantly different from value obtained in the absence of Glu. ##P < 0.01, significantly different from the value obtained in the presence of Glu alone. Values are the mean ± SE from four independent experiments.

Article Snippet: TA cloning vector, pGL3 basic vector, and Dual luciferase assay system were purchased from Promega (Madison, WI).

Techniques: Cell Differentiation, Cell Culture, Concentration Assay, Control, Transfection, Plasmid Preparation, Luciferase, Activity Assay