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Fig. 6. Reducing VASP with siRNA impairs formation of the osteoclast attachment and eliminates motility in response to NO. (A) Western blot showing reduction of VASP in cells with VASP-specific sequences versus a noncoding siRNA construct. This transfection used a mixture of four siRNAs. The knockdown efficiency (80-90%) was sufficient to show clear differences. (B) Impaired attachment with altered vß3 distribution in VASP-targeted siRNA-treated cells on bone. The siRNA was labeled with Cy3 to allow identification of transfected cells. Approximately 85% of attached cells were labelled with Cy3 (red). In contrast to the effect of PKG I knockdown, where the attachment ring was intact (Fig. 3), VASP-knockdown cells had an abnormal attachment with a fragmented vß3 ring (green). After VASP knockdown, the attachment structure did not change significantly with the cGMP agonist 8-pCPT-cGMP (100 µM, 1 hour). Each field is 40 µm horizontally. (C) Effect of a cGMP agonist on motility in mock-transfected and VASP-inhibited cells. There was no measurable motility in VASP-transfected cells when cells were exposed to 50 µM 8-pCPT-cGMP. The effect on motility was essentially the same as the effect of PKG I knockdown, but effects on the cell attachment were not observed with PKG I knockdown. Results are the means±s.d. of ten separate experiments.

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