Fig. 3. Eliminating PKG I from human osteoclasts prevents cGMP-induced motility and eliminates cGMP-dependent reorganization of the attachment ring. (A) Gene silencing at 48 hours post siRNA transfection reduced PKG I 95% by densitometry of western analysis. Real-time PCR gave a similar result (tenfold reduction, not shown). The western blot shows osteoclast lysates from cells treated with siRNA for PKG I compared to lysate from cells after mock transfection using a noncoding siRNA sequence. (B) Effect on motility in transfected cells. Means±s.d. of eight cells are shown. Time-lapse photography over 2 hours after addition of 8-pCPT-cGMP (see Fig. 1). Inset, motion over 20 frames by digital difference (lighter image, outlined in yellow, new position; darker image, outlined in green, old position). Note that the siRNA-treated cell has spread slightly, without moving, whereas the mock-transfected cell has moved about one cell diameter in this period. The variable diameter in the moving mock-transfected cell is typical of motile osteoclasts (see also Movie 1 in supplementary material). (C) Effect on cGMP-induced detachment. In each frame, a single osteoclast on bone is shown, with actin labeled with Alexa-488 phalloidin (green) and siRNA labeled with Cy3 (red). In frames 1 and 2, the transfected siRNA is specific for PKG I. The cell in frame 2 was also treated with 8-pCPT-cGMP, 100 µM for 1 hour, but its attachment ring was unaffected. The cell in frame 3 (bottom frame) was also transfected, but with a noncoding siRNA. In this case, its response to 8-pCPT-cGMP was normal, and the attachment has broken up into discrete clumps (arrows). Each field is 80 µm square.