Figure 6. Reduced proliferation and survival of floating spheres from Opg^−/− spiral ganglion.

(A) Ten spheres were picked (hence there were no error bars for the first propagation) at the third passage and propagated twice. Opg^−/− spheres proliferated slower than WT spheres. The error bars indicate the standard errors of three replicates. (B) Opg^−/− spheres were smaller than WT spheres. Scale bar: 50 μm. (C) Floating spheres from Opg ^−/− mice were significantly more apoptotic than WT spheres, as reflected in expression of cleaved caspase 3 detected by immunocytochemistry per DAPI positive nuclei in spheres. The error bars indicate the standard errors of 4 replicates. For each replicate, cells were counted in 4 randomly selected fields of each plate. (D) Counting of floating Opg ^−/− spheres treated with BrdU revealed that OPG and zoledronate treatment increased, whereas RANKL treatment had a tendency to reduce, the number of BrdU positive cells per DAPI positive nuclei in spheres. The error bars indicate the standard errors of three replicates. The fraction of BrdU+ cells was counted similar to the counting in (C). Representative images of the cells were shown in (F) – top panel. (E) Cleaved caspase 3 immunostaining showed that Rankl treatment increased whereas OPG and zoledronate treatment decreased the number of cleaved caspase 3 positive apoptotic cells per DAPI positive nuclei in spheres. The error bars indicate the standard errors of 4 replicates. The fraction of caspase 3 + cells was counted as in (C). Representative images of the cells were shown in (F) – bottom row. (F) Representative images of Opg^−/− spheres stained with DAPI to label nuclei and anti-BrdU antibody to label proliferating cells (top panel) or anti-cleaved caspase 3 antibody to label apoptotic cells (bottom panel) in response to no treatment (control), or treatment with RANKL, OPG, or zoledronate. Scale bar applies to all figures and represents 50 μm.