FIGURE 6.

Cbl^YF/YF OCLs demonstrate enhanced survival as a result of increased PI3K activity. OCLs were either untreated or were treated with RANKL (50 μg/ml) for 48 h. After treatment, cells were fixed and TRAP-stained. A, photomicrographs of the TRAP-stained OC culture with the indicated treatment. Magnification was ×10. B, histogram represents the data expressed as a percentage of the initial number of TRAP + MNCs surviving after 48 h. (gray bars, WT; black bars, Cbl^YF/YF). *, p < 0.05 as compared with WT control. C, to visualize apoptotic OCLs on day 5 of differentiation, OCLs were fixed and stained with DAPI and TUNEL. The arrows indicate bright green TUNEL-positive cells showing apoptotic nuclei. D, to determine onset of apoptosis after 5 days of culture, cells were either left untreated or treated with RANKL (50 μg/ml) for indicated times. TUNEL-positive cells were counted, and data are expressed as percentage of TUNEL-positive OCLs (gray bars, WT; black bars, Cbl^YF/YF). *, p < 0.05 as compared with WT control. E, to determine the caspase-3 activity, OCLs were either left untreated or treated with RANKL (50 μg/ml) for 3 h. Caspase activity was measured as described under “Experimental Procedures” (gray bars, WT; black bars, Cbl^YF/YF). F and G, to determine the role of AKT in the prolonged survival of Cbl^YF/YF OCLs, cells were treated with RANKL (50 μg/ml) or were treated with increasing concentrations of PI3K inhibitor LY294002 (1–10 μm) for 12 h. F, blots were probed with anti-phospho-AKT Thr³⁰⁸ (top) and anti-AKT antibodies (bottom). G, cells were fixed, and the TUNEL assay was performed as described above, and data are expressed as percentage of TUNEL-positive OCLs. *, p value between 0 and 1 or 10 mm in WT; #, p value between 0 and 10 mm in Cbl^YF/YF OCLs; ^, p value showing significance between WT and Cbl^YF/YF OCLs in the presence of RANKL. Data are representative of three independent experiments. RLU, relative luminescence units; Error bars, S.D.