Fig. 9. Defects in bone nodule formation and spheroid compaction with Icap-1^-/- osteoblasts. (A) Immortalized SV6.5 (wild-type) or SV2.1 (Icap-1^-/-) preosteoblasts were induced to differentiate in vitro for 15 days, then bone nodule formation and organization were visualized by phase contrast microscopy. The inset is a higher magnification view of the boxed region. Note that SV2.1 cells are less cohesive than control cells. (B) Spheroids were formed from SV2.1, from SV2.1 rescued with Icap-1 cDNA or from SV6.5 preosteoblasts and analyzed after 16 hours incubation at 37°C using a standard protocol for the hanging drop assay. We used 25,000 cells per drop in each experimental condition. For antibody treatment, 9EG7 or control antibodies were added at a final concentration of 10 µg/ml during the condensation process in a medium supplemented with an FN-depleted serum. Note that cellular compaction is delayed in both Icap-1^-/- and 9EG7-treated wild-type osteoblast compared with untreated wild-type cells.