Fig. 5. Distribution of PKG I, VASP and migfilin in cGMP-inhibited and cGMP-activated osteoclasts. In osteoclasts grown on glass transfected with siRNA specific for PKG 1 (A), VASP (green) was partially distributed in a peripheral ring (arrows) similar to vß3 (see Fig. 2) whereas migfilin (red) was cytoplasmic. In contrast, in cGMP-stimulated cells (B), migfilin clearly colocalized with the peripheral VASP (arrows). Migfilin distribution in cells on bone was consistent with that in A and B, but is seen better in the higher resolution possible on glass substrate, and only glass-attached cells are shown. (C) In osteoclasts on bone with the cGMP inhibitor Rp-cGMPS at 50 µM, PKG I (green) was distributed in the cytoplasm; very little PKG I occurred in the cell periphery, marked by phalloidin (red) to label actin (arrows). (D) With the addition of 100 µM 8-pCPT cGMP, a nonhydrolysable cGMP analog, PKG I and actin were redistributed and there was some overlap (arrows). In cGMP agonists at high concentrations over longer times, nuclear localization of PKG I also occurred (not illustrated); extensive nuclear labeling correlated with complete detachment and may be associated with cell death (see text). Bar, 20 µm (A,B); 10 µm (C,D).