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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.
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