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. 2011 Sep 22;286(46):39871–39881. doi: 10.1074/jbc.M111.295048

FIGURE 7.

FIGURE 7.

The effects of 11R-VIVIT, a specific inhibitor of NFAT activation, on OPN-induced nuclear translocation of NFATc1 and osteoclast survival. A, rabbit osteoclasts, suspended in supplemented M199 buffered with HEPES (25 mm) and HCO3− (26 mm) were treated with vehicle or 11R-VIVIT (VIVIT, 3 μm) for 15 min. In a separate series of experiments, cells were treated with the inactive control peptide 11R-VEET (VEET, 3 μm) for 15 min. Cells were then plated on uncoated glass (Control) or coverslips coated with OPN. After a 1-h incubation at 37 °C in 5% CO2, preparations were washed and cultures were incubated for an additional 2 h in the continued presence of vehicle, 11R-VIVIT (VIVIT, 1 μm) or 11R-VEET (VEET, 1 μm). Preparations were then fixed and NFATc1 localization was assessed by immunofluorescence. Histograms show the proportion of osteoclasts exhibiting nuclear localization of NFATc1. Data are mean ± S.E., n = 3 independent experiments, *, p < 0.01. In the left panel, data are normalized as a fraction of vehicle-treated control; in the right panel, data are normalized as a fraction of control. B, rat osteoclasts, suspended in supplemented M199 buffered with HEPES (25 mm) and HCO3− (26 mm), were treated with vehicle or 11R-VIVIT (VIVIT, 3 μm) for 15 min. In a separate series of experiments, cells were treated with 11R-VEET (VEET, 3 μm) for 15 min. Cells were then plated on uncoated glass (control) or coverslips coated with OPN. After a 1-h incubation at 37 °C in 5% CO2, preparations were washed. Survival was assessed by counting the number of osteoclasts before and after an additional 18 h of culture in the continued presence of vehicle, 11R-VIVIT (1 μm) or 11R-VEET (VEET, 1 μm). The number of surviving osteoclasts on each coverslip at 18 h was expressed as a percentage of the initial number of osteoclasts on the same coverslip. Data are mean ± S.E., n = 3 independent experiments, *, p < 0.05. In the left panel, data are normalized as a fraction of vehicle-treated control; in the right panel, data are normalized as a fraction of control.